U.S. patent application number 13/030595 was filed with the patent office on 2011-09-01 for methods of manufacture of therapeutic products comprising vitalized placental dispersions.
Invention is credited to Alla Danilkovitch, Timothy Jansen, Samson Tom, Dana Yoo, Jaime Zerhusen.
Application Number | 20110212065 13/030595 |
Document ID | / |
Family ID | 44476694 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212065 |
Kind Code |
A1 |
Jansen; Timothy ; et
al. |
September 1, 2011 |
METHODS OF MANUFACTURE OF THERAPEUTIC PRODUCTS COMPRISING VITALIZED
PLACENTAL DISPERSIONS
Abstract
This invention provides a fluid therapeutic placental product
comprising placental cells and a placental dispersion comprising
placental factors. The placental cells and the placental dispersion
are derived from placental tissue. A placental tissue can
optionally be an amnion, chorion, or a trophoblast-depleted
chorion. 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: |
Jansen; Timothy; (Baltimore,
MD) ; Tom; Samson; (Baltimore, MD) ;
Danilkovitch; Alla; (Columbia, MD) ; Yoo; Dana;
(Sykesville, MD) ; Zerhusen; Jaime; (Columbia,
MD) |
Family ID: |
44476694 |
Appl. No.: |
13/030595 |
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: |
424/93.7 |
Current CPC
Class: |
A01N 1/0221 20130101;
A61K 35/50 20130101; C12N 2501/115 20130101; A61K 38/1825 20130101;
A61P 17/00 20180101; C12N 2502/025 20130101; C12N 5/0605 20130101;
A61K 38/39 20130101; A61K 35/28 20130101; A61K 38/1841 20130101;
C12N 2500/02 20130101; A61K 38/57 20130101; A61P 17/02 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/50 20060101
A61K035/50; A61P 17/02 20060101 A61P017/02 |
Claims
1-36. (canceled)
37. A method of manufacturing a therapeutic placental product
comprising: a) obtaining a first placental tissue; b) obtaining
placental cells from the first placental tissue; c) obtaining a
second placental tissue; d) disrupting the second placental tissue
to form a dispersion comprising placental factors; and e) combining
the placental cells and the placental dispersion to form the
placental product, wherein the placental dispersion is a
homogenate.
38. The method of claim 37, wherein the first placental tissue is
autologous to the second placental tissue and wherein the first
placental tissue and the second placental tissue are chorionic
tissue.
39. The method of claim 38, wherein the second placental tissue is
derived from the fist placental tissue after the step of obtaining
the placental cells.
40. The method of claim 39, wherein the step of obtaining the
placental cells comprises exposing the first placental tissue to a
protease, optionally wherein the protease is a collagenase.
41. The method of claim 40, wherein the protease exposing step has
a duration of about 30 minutes or less.
42. The method of claim 40, wherein the protease exposing step
releases less than about 10% of the maximum number of releasable
placental cells.
43. The method of claim 38, wherein the chorionic tissue is
depleted of trophoblasts.
44. The method of claim 43, wherein the trophoblasts are depleted
by dissection and dispase treatment.
45. The method of claim 37, wherein the placental product comprises
one or more members selected from the group consisting of
extracellular matrix proteins; protease inhibitors; angiogenic
factors; and placental factors which promotes the migration of
epithelial cells into a wound.
46. The method of claim 37, wherein the placental product comprises
one or more protease inhibitors selected from the group consisting
of matrix metalloproteinases (TIMPs), alpha-2 macroglobulin, and
thrombospondins.
47. The method of claim 37, wherein the placental product comprises
one or more angiogenic factors selected from the group consisting
of VEGF and bFGF.
48. The method of claim 37, wherein the placental product comprises
one or more factors which promote the migration of epithelial cells
into a wound selected from the group consisting of HGF and KGF.
49. The method of claim 37, wherein the placental cells are
selected from the group consisting of MSCs, ESCs, placenta-derived
mesenchymal progenitor cells, placental mesenchymal stem cells
fibroblasts, epithelial cells, placental mesenchymal cells, and
macrophages.
50. The method of claim 49, wherein the placental cells are present
at a concentration of at least about 20,000 per ml of placental
product.
51. The method of claim 37, wherein at least one of the placental
cells or the placental dispersion is cryopreserved.
52. The method of claim 37, wherein the placental cells are
cryopreserved before combining with the placental dispersion, and
wherein the placental dispersion is optionally cryopreserved before
combining with the placental cells.
53. The method of claim 37, wherein the placental cells comprise
stromal cells and the placental cells are cryopreserved.
54. The method of claim 39, wherein: a. the step of obtaining the
placental cells comprises exposing the first placental tissue to a
protease; b. the protease exposing step is of about 30 minutes or
less duration; c. the protease exposing step releases less than
about 10% of the maximum number of releasable placental cells; d.
the chorionic tissue is depleted of trophoblasts; e. the placental
product comprises one or more protease inhibitors selected from the
group consisting of matrix metalloproteinases (TIMPs), alpha-2
macroglobulin, and thrombospondins; f. placental product comprises
one or more of VEGF and bFGF; g. the placental product comprises
one or more of HGF and KGF; h. the placental cells comprise MSCs
and cells selected from the group consisting of ESCs,
placenta-derived mesenchymal progenitor cells, placental
mesenchymal stem cells fibroblasts, epithelial cells, placental
mesenchymal cells, and macrophages; i. the placental cells are
present at a concentration of at least about 20,000 per ml of
placental product; and j. at least one of the placental cells and
the placental dispersion is cryopreserved.
55. A method of treating a tissue injury comprising administering
to a subject in need thereof a therapeutic placental product made
by the method of claim 37.
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]
"Immunocompatible Chorionic Membrane Products", [0007] "Methods of
Manufacture of Immunocompatible Chorionic Membrane" Products,
[0008] "Immunocompatible Amniotic Membrane Products", [0009]
"Methods of Manufacture of Immunocompatible Amniotic Membrane
Products", and [0010] "Therapeutic Products Comprising Vitalized
Placental Dispersions".
TECHNICAL FIELD
[0011] The present invention relates to placental products, methods
of medical treatment using placental products, and methods of
making placental products.
BACKGROUND
[0012] The structural integrity of tissue is achieved, in part, by
a dynamic interaction of the tissue with bioactive molecules,
extracellular matrix, and a host of circulating cell types. Such
interactions are also pivotal during tissue aging, injury,
restorative and regenerative treatments. For example, burns produce
local tissue damage as well as systemic consequences. Currently,
treatment of burn wounds is focused on promoting healing and
decreasing the risk of infection. Burn wounds continue to be a
frustrating and serious problem in the clinic, and these wounds are
often accompanied by high morbidity and mortality rates. The
standard of care for burns includes the use of antiseptics and
gauze wound dressings. However, for severe and large surface area
burns, this treatment is not satisfactory. The gold standard for
severe burn treatment continues to be autologous living skin
grafts. However, the amount of skin available for grafting is often
extremely limited, and this procedure always results in donor site
wounds.
[0013] Attempts to improve burn wound care have included the use of
a single growth factor or cocktail of growth factors as well as
biological skin substitutes. Growth factors such as epidermal
growth factor (EGF), platelet derived growth factor (PDGF), basic
fibroblast growth factor (FGF), vascular endothelial growth factor
(VEGF), and other singular factors have been tested in burn wound
healing; however, with varying results.
[0014] The use of placental membranes for burns and other types of
wounds originated more than 100 years ago (reviewed by Kesting et
al., 2008). Placental membranes contain components that are present
in skin and required for wound healing such as extracellular
matrix, growth factors, and cells, including MSCs that are
responsible for orchestrating the process of wound healing. The
effectiveness of placental membranes such as amniotic membranes for
burns was recorded in a number of published reports; however, the
use of placental membranes for large surface area burns is limited
due to challenges in providing sufficient placental membranes to
cover large areas.
[0015] What is needed in the art is a therapeutic product that
provides the benefits of placental membranes yet can be applied in
fluid form. Moreover, needed is a product that provides dynamic
therapy throughout more than one, optimally all, of the phases of
wound repair: inflammatory, proliferative, and remodeling.
SUMMARY OF THE INVENTION
[0016] The present invention provides methods of manufacturing
placental products comprising placental cells and a placental
dispersion comprising placental factors. The placental cells and
the placental dispersion are derived from placental tissue, e.g. a
whole placenta or portion thereof. Placental tissue can be obtained
by mechanical manipulation (e.g. dissection) or enzymatic digestion
or combinations thereof. A placental tissue can optionally be an
amnion, chorion, a mixture of amnion and chorion, or other tissue
described here.
[0017] The present invention also provides a method of treating a
tissue injury (e.g. wound or burn) comprising administering to a
patient in need thereof a placental product of the present
invention.
[0018] Optionally, the placental dispersion is a homogenate.
[0019] Optionally, placental factors present include extracellular
matrix components.
[0020] Optionally, the placental dispersion comprises one or more
placental factors set forth in Table 1, Table 2, Table 3, or Table
5.
[0021] Optionally, the placental cells comprise stromal cells such
as MSCs (mesenchymal stem cells) and PSCs (placental stem
cells).
[0022] In one embodiment, the method of making a placental product
is a parallel processing method that comprises: [0023] i) obtaining
a first placental (e.g. amniotic or chorionic) tissue; [0024] ii)
obtaining placental cells from the first placental tissue; [0025]
iii) obtaining a second placental (e.g. amniotic or chorionic)
tissue; [0026] iv) disrupting the second placental tissue to form a
dispersion comprising placental factors; [0027] v) combining the
placental cells and the dispersion to form the placental
product.
[0028] Optionally, the first placental tissue and the second
placental tissue are autologous to each other, for example, derived
from the same donor.
[0029] In one embodiment, the method of making a placental product
is a serial processing method wherein the second placental tissue
is derived from the first placental tissue after said step of
isolating the placental cells from a first placental tissue. For
example, a first chorionic tissue may be retained after isolating a
population of cells thereof, and then disrupted to form a
dispersion. The dispersion may then be combined with the placental
cells.
[0030] Optionally, the step of isolating the placental cells
comprises contacting the first placental tissue (e.g. amnion or a
chorion or a chorion lacking trophoblasts) with a digestive enzyme,
such as a collagenase II. Optionally, the first placental tissue is
exposed to a limited digestion with an enzyme such as collagenase
II; e.g. exposure for less than about 1 hour (e.g. about 10 minutes
or about 20 minutes).
[0031] Optionally, the placental tissue (from which the placental
dispersion is produced) is chorionic tissue depleted of
trophoblasts by treatment with a digestive enzyme such as dispase
II followed by physical removal.
[0032] In another embodiment, the method of making a placental
product comprises: [0033] i) obtaining a placental (e.g. amniotic
or chorionic) tissue; [0034] ii) exposing the placental tissue to
collagenase; [0035] iii) dividing the placental tissue into a first
portion and a second portion; [0036] iv) isolating placental cells
from the first placental portion; [0037] v) disrupting the second
placental portion to form a dispersion comprising placental
factors; and [0038] vi) combining the placental cells and the
placental dispersion to form the placental product. [0039] vii) In
another embodiment, the method of making a placental product
comprises: [0040] viii) obtaining a placental (e.g. amniotic or
chorionic) tissue; [0041] ix) exposing the placental tissue to a
collagenase for a time sufficient to release placental cells;
[0042] x) isolating the released placental cells from the
collagenase exposed placental tissue; [0043] xi) disrupting the
collagenase exposed placental tissue to form a dispersion
comprising placental factors; and [0044] xii) combining the
placental cells and the placental dispersion to form the placental
product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 depicts cell viability, before and after a
freeze-thaw cycle of a placental product comprising isolated cells
and a placental dispersion.
[0046] FIG. 2 depicts recovery of viable cells isolated by
digestion.
[0047] FIG. 3 depicts cell viability, before and after a
freeze-thaw cycle of a placental product comprising isolated cells
and a placental dispersion.
[0048] FIG. 4 depicts recovery of viable cells isolated by
digestion.
[0049] FIG. 5 depicts the level of viable cells in a placental
product made with or without a step of cell isolation before tissue
disruption.
[0050] FIG. 6 depicts cell phenotype of cells in a placental
product.
[0051] FIG. 7 depicts cell viability using various
cryoprotectants
[0052] FIG. 8 depicts placental tissue weight and live cells
recovered following collagenase treatment of various incubation
times.
[0053] FIG. 9 depicts the number of collagenase-released cells from
multiple donors.
[0054] FIG. 10 depicts viable cell level in a placental product
when a placenta is subjected to hypoxic or normoxic conditions.
[0055] FIG. 11 depicts cell viability when a placenta is subjected
to hypoxic or normoxic conditions.
[0056] FIG. 12 depicts expression of bFGF in placental products for
14 days in culture.
[0057] FIG. 13 depicts expression of IFN-2.alpha. and TGF-.beta.3
in placental products,
[0058] FIG. 14 BMP-2, BMP-4, BMP-7, PLAB, PIGF, and IGF-1 were
detected in placental products derived from the chorionic
membrane
[0059] FIG. 15. Representative image of passage 2 cells isolated
and expanded from a placental product derived from the chorionic
membrane
[0060] FIG. 16 depicts recovery of viable cells isolated by
digestion using various collagenase II enzymes.
[0061] FIG. 17 depicts cell viability, before and after a
freeze-thaw cycle of a placental product comprising isolated cells
and a placental dispersion.
DETAILED DESCRIPTION OF THE INVENTION
[0062] As used here, the following definitions and abbreviations
apply. [0063] "Chorionic tissue" or "Chorionic membrane" means the
chorion or a portion thereof, e.g. the trophoblast, the somatic
mesoderm, or combinations thereof. [0064] "Examplary" (or "e.g." or
"by example") means a non-limiting example. [0065] "Placental
dispersion" means a product formed by physical/mechanical
disruption of placental tissue. For example, a dispersion may be in
the form of a homogenate, a blend, a suspension, a colloid, or a
solution. [0066] "Placental tissue" means tissue derived from the
placenta in the broadest sense of the word. Placental tissue can be
a whole placenta or any portion thereof. "Portions of the placenta"
is meant to include chorion, amnion, a chorion and amniotic
membrane (e.g. amnio-chorion), Wharton's jelly, umbilical cord,
placental cotyledons or combinations thereof. The placental tissue
may be dissected or digested (or combinations thereof) to remove
portions, membrane, or structures. [0067] "Placental cells" means
any cell that can be obtained from a placenta, without regard to
genetic origin (e.g. maternal vs. fetal), developmental origin
(e.g. endodermal, ectodermal, or mesodermal), or differentiation.
Placental cells may comprise any placental cells known in the art,
for example, mesenchymal stem cells (MSCs), endometrial stromal
cells (ESCs), placenta-derived mesenchymal progenitor cells,
placental mesenchymal stem cells, fibroblasts, epithelial cells,
placental mesenchymal cells, macrophages, and the like. [0068]
"Placental cells" are further meant to require some feature of live
cells such as one or more of metabolic activity, structural
integrity (e.g. exclusion of a viability stain such as methylene
blue), mitotic activity, signal transduction, and the like. [0069]
"Placental factor" means any product that is obtainable from a
placental tissue (or placental cells). The product can be an
angiogenic factor, chemokine, cytokine, growth factor, protease,
protease inhibitor, or matrix component. Examplary placental
factors are listed in Table 1, Table 2, Table 3, and Table 5.
[0070] "Tissue injury" means an injury of any tissue such as skin
or the outer layer of any organ. By injury, it is meant a pathology
that involves or results from an mechanical, metabolic or other
insult. Examples of such tissue injuries are burns, wounds,
ulcerations, and lacerations, ablations (including laser, freezing,
cryo-surgery, heat and electrical ablations), and surgical
incisions.
Placental Product
[0071] Overview
[0072] It has been surprisingly discovered that a placental product
can now be produced by combining placental cells and a placental
dispersion to produce a medicinal product of substantial and
superior therapeutic value when administered to a tissue injury.
The placental product has several advantageous properties.
[0073] Fluidity. The placental product shares certain properties of
a fluid such as an ability to deform under an applied stress and
can be quantified measurements of viscosity. Thus, the present
placental product can be spread over the surface of the surface to
which it is applied. For example, one ml of placental product can
be spread topically to cover more than about any of about 1
cm.sup.2, about 10 cm.sup.2, about 25 cm.sup.2, about 50 cm.sup.2,
or about 100 cm.sup.2 of skin. This fluid property solves the
problem of limited applicability of products that retain the
non-elastic properties of tissue (e.g. skin grafts). Moreover, the
fluidity of the present placental product now makes it practical
for new uses such as application to articulating joints and curved
surfaces. It also provides a means of rapid application.
[0074] Extended release. Extended release formulations, especially
for topical pharmaceutical products, are especially problematic.
Moreover, due to natural instabilities as well as metabolic
degradation, topical formulations often exhibit substantial loss of
activity with time after administration. Without being bound by
theory, the inventors believe that the placental cells of the
present placental products produce placental components after
administration. Thus, the present placental products can contain
placental components derived from the placental dispersion and
derived from the placental cells and depletion of placental
components can be reduced. Additionally, placental cells in the
present placental product can produce placental factors (e.g.
protease inhibitors) that reduce the metabolic degradation of
placental factors.
[0075] Dynamic responsivity. Without being bound by theory, the
inventors believe that presence of live placental cells provide to
the placental product the capacity to respond to physiologic
stimuli in a manner somewhat analogous to endogenous cells in situ.
Evidence of dynamic responsivity includes stimulated release of
placental factors or changes in the placental factor profile with
time after administration.
[0076] Placental Cells
[0077] Placental cells may be obtained from any placental tissue
(e.g. chorion). Placental cells may be obtained by processing
placental tissue in any manner which retains cell viability of at
least one cell type (e.g. MSCs). For example, placental cells may
be isolated or purified from placental tissue (e.g. by collagenase
digestion of the chorion) or may be obtained without isolation from
one or more placental factors (e.g. extracellular matrix) or from
other placental cells.
[0078] Placental cells may be obtained by any method known in the
art. Useful methods of obtaining placental cells (e.g. chorionic
cells) are described, for example, 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").
[0079] In one embodiment, placental cells are obtained by
contacting placental tissue with one or more digestive enzymes, for
example, by immersing placental tissue (e.g. a chorion, or
placental tissue lacking trophoblasts) in a solution containing the
digestive enzyme. The digestive enzyme may be any digestive enzyme
known in the art. The digestive enzyme may also be combination of
enzymes. Examplary digestive enzymes include one or more:
collagenases (e.g., collagenase I, II, Ill and IV), matrix
metalloprotease, neutral proteases, papains, deoxyribonucleases,
serine protease (e.g. trypsin, chymotrypsin, elastase), or any
combination thereof.
[0080] In one embodiment, placental cells are obtained from a
chorion by contacting a chorion (e.g. a chorion lacking
trophoblasts) with a collagenase (e.g. collagenase II). The
collagenase may present in any suitable concentration, for example,
about 100 U/mL to about 1000 mL, and in any suitable collagenase
solvent, such as DMEM, and at any suitable temperature, for example
37.degree. C. The chorion may be contacted with the digestive
enzyme for any suitable period of time. Optionally, the chorion is
contacted with a collagenase (e.g. collagenase II) for less than
about any of: about 3 hrs, about 2 hr, or about 1 hr. Optionally,
the chorion is contacted with the collagenase (e.g. collagenase II)
for less than about 1 hour, for example, less than about any of:
about 60 min, about 50 min, about 40 min, about 30 min, about 20
min, about 15 min, about 10 min, or about 5 min. Optionally, the
chorion is contacted with a collagenase for a limited period of
time such that a substantial portion of the placental tissue is
retained on a about 100 micron filter. Optionally, the chorion is
contacted with collagenase II for a limited period of time such
that a substantial portion of the placental tissue is retained on a
100 micron filter. Optionally, after the placental cells are
obtained, the chorion is disrupted to form a dispersion and the
population is combined with (e.g. added to) the dispersion.
[0081] Surprisingly, a step of obtaining placental cells before
subjecting the placental tissue to tissue disruption results in
substantially a greater number of cells generally and also results
in a population of cells that more resemble the population in the
placental tissue than population of cells that are obtained from
disrupted placental tissue.
[0082] A placental product that comprises placental cells from
placental tissue that has not been disrupted surprisingly provides
a therapeutically effective amount of viable cells without the need
for ex vivo expansion of the placental cells. Although ex vivo
expansion is a known method of increasing the number of viable
cells in a population, such a step often leads to changes in the
population make-up or distribution of cell phenotype. For example,
various cells in a population may expand at different rates and
expansion may also induce differentiation. Accordingly, one
embodiment of the present invention provides a placental product
comprising placental cells derived from a placental tissue wherein
the placental cells exhibits a phenotypic distribution of cells
which is substantially similar to the cells of the placental tissue
of origin.
[0083] Placental Dispersion
[0084] A placental dispersion may be provided by disrupting a
placenta (e.g. a chorion). The disruption of placental tissue may
be accomplished by any physical/mechanical method of disrupting
tissue (i.e. use of a "tissue disruptor" or "means for
disruption"). For example, disruption may comprise homogenization,
maceration, use of a blender, crushing, or mincing. Disruption may
additionally or alternatively comprise shearing, mincing, dicing,
or chopping. Disruption may additionally or alternatively comprise
sonication.
[0085] The placental tissue may be disrupted for any suitable
duration which produces a dispersion from the placenta. For
example, the placenta may be disrupted (e.g. homogenized) for less
than about 20 sec, about 15 sec, about 10 sec, or about 5
seconds.
[0086] The placental tissue can be disrupted sufficient to form a
placental product with fluid characteristic and yet retain viable
cells. Accordingly, live cells in the placental products of the
present invention can additionally comprise placental cells that
are derived from the placental dispersion.
[0087] The extent of tissue disruption may be reduced by a prior
enzymatic digestion step with a matrix degrading enzyme such a
collagenase(s), a protease(s), or combinations thereof. Indeed, it
has surprisingly been discovered that such prior digestion
preserves viable cells in the placental dispersion. For example,
the length of treatment by a tissue disruptor can be reduced by
prior enzymatic digestion.
[0088] Placental Factors
[0089] A placental product of the present invention may comprise
one or more placental factors where the placental factors are
components of the placental dispersion or components released into
the placental product by the placental cells or a combination
thereof.
[0090] It has surprisingly been discovered that the content of
placental factors in placental products made according to the
present invention have an unexpected therapeutic value. Such
content of placental factors as taught herein is accordingly
referred to here as a "therapeutic profile".
[0091] In one embodiment of the present invention, a therapeutic
profile is one that provides two or more, or three or more, or four
or more placental factors listed in Table 1, Table 2, Table 3, or
Table 5. Optionally, the placental factors are present in an amount
of about 20% to about 500% of the mean concentration set forth in
Table 1, Table 2, or Table 5. Optionally, the placental factors are
present in an amount of about 20% to about 500% of the minimum and
the maximum (respectively) of the values set forth in Table 1,
Table 2, or Table 5
[0092] Placental factors, according to the present invention, can
be placental-derived factors such as angiogenic factors,
chemokines, cytokines, growth factors, matrix metalloproteases,
extracellular matrix proteins (or "matrix proteins"), and
combinations thereof. The present placental products can comprise
any of these placental factors.
[0093] The present placental products can optionally comprise a
therapeutic profile of one or more of a PDGF (e.g. PDGF-bb), EGF,
FGF, TGF-.beta.1, TGF-.beta.3, and VEGF and/or one or more of IL-8,
IL-6, and MCP-1.
[0094] Useful placental products of the present invention can have
a therapeutic profile as set forth in Table 1, Table 2, Table 3, or
Table 5.
[0095] Useful placental products of the present invention can have
a therapeutic profile comprising at least 25% of the minimum
concentration of one or more placental factors set forth in Table 1
and optionally no more than 400% of the maximum concentration of
one or more placental factors set forth in Table 1. In one
embodiment, the one or more placental factors comprise fibronectin,
TIMP, TGF.beta.1, bFGF, and MMPs (e.g. MMP1, 2, 4, 7, 8, 9, and
10).
[0096] Useful placental products of the present invention can have
a therapeutic profile comprising four or more placental factors
where at least two placental factors are extracellular matrix
components (or fragment thereof).
[0097] Placental products of the present invention can comprise a
therapeutic profile of one or more placental factors which promote
the migration of epithelial cells into a wound area (e.g. HGF
and/or KGF), optionally in combination with a growth factor such as
TGF-.beta.1. Optionally the concentration of such placental factors
is about 25% of the minimum values set forth in Table 1 and
optionally no more than 400% of the maximum concentration set forth
in Table 1
[0098] Placental products can comprise a therapeutic profile of
placental factors that are mitotic or growth promoting. Placental
products can contain HGF and KGF. For example, HGF at a
concentration of about 5,000 to about 200,000 pg/mL and KGF at a
concentration of about 5,000 to about 400,000 pg/mL are present in
an examplary placental product as detailed in Example 10.
Optionally, such placental products are useful in preventing
scaring or a useful therapy aid during re-epithelialization,
[0099] Placental products of the present invention can comprise a
therapeutic profile of placental factors comprising one or more
angiogenic factors (e.g. VEGF and/or bFGF) and can optionally
additionally comprise one or more growth factors (e.g. TGF-.beta.1
and/or TGF-.beta.2),
[0100] Examplary placental products of the present invention
contain a therapeutic profile of VEGF levels greater than about 10
pg/ml or greater than about 50 pg/ml or greater than about 100
pg/ml. For example, an examplary placental product can comprise
greater than about 200 pg/ml as detailed in Example 10.
[0101] Examplary placental products of the present invention
contain a therapeutic profile of bFGF levels greater than any of
about 10 or 100 or 1,000 or 10,000 pg/ml. An examplary placental
product can comprise greater than about 11,000 pg/mL, as detailed
in Example 10. Optionally such FGF-comprising placental products
are useful for burn wound healing.
[0102] Placental products of the present invention can comprise a
therapeutic profile of TGF-.beta.1 and TGF-.beta.2. An examplary
placental product, as detailed in Example 10, comprises bFGF,
TGF-.beta.1 and TGF-.beta.2. Optionally, such placental products
are useful when the skin pathology being treated involves an
inflammatory or a scaring pathology.
[0103] Placental products of the present invention may comprise a
therapeutic profile of one or more protease inhibitors, such as
tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-2
macroglobulin, and/or thrombospondins.
[0104] In one embodiment, a placental product (e.g. derived from
chorion) comprises one or more protease inhibitors.
[0105] In one embodiment, a placental product (e.g. derived from
chorion) comprises one or more protease inhibitors and
extracellular matrix proteins
[0106] In one embodiment, a placental product (e.g. derived from
chorion) comprises one or more protease inhibitors and viable
cells.
[0107] In one embodiment, a placental product (e.g. derived from
chorion) comprises one or more protease inhibitors, extracellular
matrix proteins, and viable cells.
[0108] Without being bound by theory, the present inventors believe
that the surprising efficacy that characterizes placental products
of the present invention result in an interaction between the
placental cells and the placental factors comprising (1) growth
factor(s), (2) protease inhibitor(s), and (3) extracellular matrix
components. Growth factors can bind to extracellular matrix thereby
protecting the growth factors from degradation and effectively
extending the half life of the growth factors. Bioavailability can
be further regulated by subsequent release or matrix degradation.
Similarly, protease inhibitors in examplary placental products
provide additional protection against protease degradation. The
placental cells further can protect growth factors and other
placental factors in the placental products from degradation by
providing additional protease inhibitors and growth factors.
Accordingly, such placental products can optionally maintain
surprising product integrity for extended periods of time resulting
in placental products that require less frequent applications and
superior treatment of tissue injuries such as burns and wounds.
Surprisingly, the growth factors in such placental products can
demonstrate a longer half-life in comparison to other growth factor
therapies such as ACCS.
Formulation
[0109] The placental products of the present invention are
administered as a dermatologically acceptable pharmaceutical
product. Optionally, active pharmaceutical ingredients or
excipients or combinations thereof can be added.
[0110] Viscosity. Viscosity values that are useful and desirable
according to the present invention vary as a function of the
indication being treated. For example, where broad coverage (i.e.
large areas of skin) or lower concentrations of placental products
are desired, a less viscous formulation is advantageous. Examples
of less viscous formulations are those of about 1,000 cps to about
50,000 cps, or about 2,000 cps to about 25,000 cps, or about 2,000
cps to about 10,000 cps, or about 5,000 cps to about 15,000 cps.
Such less viscous compositions facilitate spreading of applied
composition.
[0111] Where more restricted coverage or higher levels of placental
products are desired, a more viscous formulation is advantageous.
Examples of more viscous formulations are about 20,000 cps to about
200,000 cps or about 50,000 cps to about 100,000 cps.
[0112] The skilled artisan will now readily recognize that the
desired viscosity can be attained according to the present
invention by adjustments of the dispersion method (discussed
elsewhere herein) or by selection of a dermatologically acceptable
thickening agent and empirically determining the concentration
necessary to achieve the desired thickening agent.
[0113] The placental products of the present invention can
optionally include one or more antibiotic, emollient, keratolytics
agent, humectants, anti-oxidants, preservative, or combinations
thereof.
[0114] In one embodiment, a placental product comprises albumin,
such as HSA or BSA. Optionally, the placental product comprises an
electrolyte solution, for example, to provide physiological
osmolality and pH (e.g. Plasma-LyteA). Optionally, the placental
product comprises a cryopreservative, such as DMSO, glycerol,
sericin, sugars, or a mixture thereof.
[0115] In one embodiment, a placental product comprises albumin, an
electrolyte solution, and a cryopreservative. Optionally, the
therapeutic product comprises 1% to about 15% albumin by weight and
about 5% to about 20% cryopreservative by volume (e.g. about 10%).
Optionally, the albumin is HSA, the electrolyte solution is
Plasma-Lyte A, and the cryopreservative is DMSO.
Manufacture
[0116] Overview
[0117] A placental product of the present invention may be
manufactured from a placenta in any suitable manner that provides
the technical features taught herein. Any placental tissue is
useful according to the present invention. Each of the embodiments
of the present invention set forth here are meant to specifically
embrace placental products where the placental dispersion is a
dispersion of chorion that is depleted of or lacking trophoblastic
components.
[0118] According to the present invention, the placenta is
processed to produce the placental dispersion and the placental
cells.
[0119] In one embodiment, the placental dispersion and the
placental cells are derived from a different placenta or different
placental portion (e.g. parallel processing).
[0120] In one embodiment, the placental dispersion and the
placental cells are derived from the same placenta or the same
placental portion (e.g. sequential processing).
[0121] Manufacturing Method 1. In one embodiment, a placental
product is manufactured by: [0122] obtaining a placental (e.g.
chorionic) tissue; [0123] digesting the placental tissue with one
or more matrix degrading enzymes (e.g. a collagenase, optionally
collagenase II); [0124] obtaining placental cells from the digested
placental tissue; [0125] disrupting the digested placental tissue
with a tissue disruptor to form a placental dispersion comprising
placental factors; and [0126] combining the placental cells and the
placental dispersion to form the placental product.
[0127] Optional Manufacturing Method 2 In one embodiment, a
placental product is manufactured by: [0128] obtaining a first
placental (e.g. chorionic) tissue; [0129] digesting the first
placental tissue with one or more matrix degrading enzymes (e.g. a
collagenase, optionally collagenase II); [0130] obtaining placental
cells from the digested first placental tissue; [0131] obtaining a
second placental tissue; [0132] disrupting the second placental
tissue with a tissue disruptor to form a placental dispersion
comprising placental factors; and [0133] combining the placental
cells and the placental dispersion to form the placental
product.
[0134] For either Manufacture Method, the placental tissue can be a
chorion tissue such as a chorion tissue that has been processed to
reduce the number of trophoblastic cells.
[0135] Examplary placental products of the present invention can be
manufactured or provided with a bandage or wound dressing.
[0136] Trophoblast Removal
[0137] In one embodiment, trophoblasts are depleted or removed to
produce the placental tissue from which the placental cells or the
placental dispersion or both are derived. Surprisingly, such a
placental product has one or more of the following superior
features: [0138] a. is substantially non-immunogenic; [0139] b.
provides remarkable healing time; and [0140] c. provides enhanced
therapeutic efficacy.
[0141] Trophoblasts may 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, the trophoblasts are removed
before isolating a population of cells and/or disrupting the
placental tissue.
[0142] 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 scraping. Optionally, scraping
comprises scraping with a soft instrument such as a finger.
[0143] 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").
[0144] Preservation
[0145] A placental product of the present invention may be used
fresh or may be preserved for a period of time.
[0146] Also as depicted in FIG. 1, a placental product of the
present invention, cell viability is retained surprisingly well
after a freeze-thaw cycle
[0147] In one embodiment, a placental product is cryopreserved. A
placental product may be cryopreserved by freezing (e.g. a
-80.degree. C.). Freezing may comprise storage in a
cryopreservation medium such as DMSO, glycerol, sericin, sugars, or
mixtures thereof. Freezing may 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.
[0148] A placental product may be formulated in a cryopreservative
before cryopreservation. Examplary cryopresevatives include DMSO,
glycerol, and the like. The cryopreservative may further be
formulated with additional components such as albumin (e.g. HSA or
BSA), an electrolyte solution (e.g. Plasma-Lyte), or a combination
thereof. Optionally, the placental product comprises 1% to about
15% albumin by weight and about 5% to about 20% cryopreservative by
volume (e.g. about 10%).
[0149] Optionally, a placental product can be formed by the
addition of cryopreserved placental cells of the present invention
to a fresh (never frozen) placental dispersion or to a frozen
placental dispersion or to a lyophilized placental dispersion.
[0150] Optionally, a placental product can be formed by the
addition of fresh placental cells of the present invention to a
frozen placental dispersion or to a lyophilized placental
dispersion.
Methods of Use
[0151] The placental products 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.
[0152] 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.
[0153] Placental products can be administered autologously,
allogeneically or xenogeneically.
[0154] 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.
[0155] In one embodiment, the injury is 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.
[0156] In one embodiment, the injury is an ulcer, for example, a
diabetic ulcer (e.g. foot ulcer).
[0157] 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.
[0158] In one embodiment, a placental product is administered to
the epidermis to reduce rhytids or other features of aging skin.
Such treatment is also usefully combined with so-called cosmetic
surgery (e.g. rhinoplasty, rhytidectomy, etc.).
[0159] 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).
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] In one embodiment, a placental product of the present
invention is used to reduce fibrosis by applying the placental
product to a wound site.
[0169] 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).
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] The citations provided herein are hereby incorporated by
reference for the cited subject matter.
[0175] In the present specification, use of the singular includes
the plural except where specifically indicated.
EXAMPLES
Example 1
Obtaining Placental Tissue
[0176] A whole placenta was obtained from a registered tissue bank
after informed consent. The placenta and placed, with the maternal
surface (rough surface) face down, on a sterile tray. The
amniotic-chorionic membrane was cut and removed from the placenta.
The chorionic membrane was then separated from the amnion and
washed twice in PBS.
[0177] The chorionic membrane was then soaked in an anticoagulant
(ACD-A) solution to remove blood clots and then washed again in
PBS.
[0178] The chorionic membrane was then digested by incubation with
dispase II for 30 min. at 37.degree. C. The trophoblast layer was
mechanically removed by scraping with fingers and the chorion was
washed again in PBS.
[0179] The chorionic membrane was then incubated for 24 hours in an
antibiotic cocktail containing gentamicin, vancomycin, and
amphotericin B, and washed again in PBS.
Example 2
Digesting Placental Tissue
[0180] A chorion membrane (obtained from Example 1) was digested by
incubation in 200 mL of a collagenase II solution (300 U/mL in
DMEM) for 10 min at 37.degree. C. The chorionic membrane was then
removed, leaving a digestion suspension containing collagenase and
placental cells.
[0181] The volume and container for digestion was determined based
on the need to provide a suitable digestion environment for the
tissue once placed on a shaker. The digestion was carried out on a
standard plate shaker set at moderate speed in a 37.degree. C. cell
culture incubator.
Example 3
Obtaining Placental Cells
[0182] A digestion suspension comprising placental cells (obtained
from Example 2) was centrifuged at 2000 rcf for 5 min to separate
the digestive enzyme (collagenase II) from the placental cells.
This step centrifugation step may enhance cell viability by
preventing over-digestion and ensure that the enzyme is washed away
before homogenizing the tissue. This centrifugation step pellets
the cells without damaging them, allowing the collagenase II to be
removed as supernatant.
[0183] The cells were then centrifuged again, the supernatant
poured off, and the placental cells were resuspended in a small
volume (2 mL) of cryprotectant (5% DMSO in saline). Two mL provides
an adequate volume to resuspend the cells while not over-diluting
the chorion membrane dispersion once the cells have been added.
Example 4
Obtaining a Placental Dispersion
[0184] A chorionic membrane (obtained from Example 2) was washed
twice in PBS to remove residual digestion enzyme and placed in a
homogenization container with 1 ml cryoprotectant per gram of
chorionic membrane. This volume was determined to be appropriate
for diluting the chorion membrane enough to produce a dispersion of
ideal consistency while maintaining protein concentration at
clinically significant levels.
[0185] The temperature of the chorionic membrane was reduced by
placing the container on ice for greater than 10 min. The chorionic
membrane was then homogenized twice at high speed for 5 sec. using
a tissue homogenizer to obtain a chorionic dispersion
(homogenate).
[0186] Once the chorion membrane is subjected to digestion, it
becomes easy to homogenize. Surprisingly, only a small amount of
homogenization is needed to create a homogenous solution ideal for
clinical use and increases the amount of live cells present in the
final dispersion.
Example 5
Providing a Placental Product
[0187] A placental dispersion (obtained from Example 4) was
combined with viable isolated placental cells (obtained from
Example 3) and mixed thoroughly to provide a placental product. The
placental product may be used (e.g. for therapy) fresh or may first
be preserved (e.g. cryogenically) for a period of time.
Example 6
Cryopreservation
[0188] A placental product (obtained from Example 5) was aliquoted
into vials and incubated at 4.degree. C. for 30-60 min. The vials
were then frozen at -80.degree. C. until use.
Example 7
Isolation of Cells without Complete Digestion of Placenta
[0189] The inventors tested whether a limited collagenase II
digestion might be performed to obtain a suspension containing live
cells and yet preserve the integrity of the placental tissue (e.g.
preserve placental factors and remaining live cells). A brief 10
minute digestion with collagenase II left the tissue intact and
made further handling possible. In addition, a 10 min. collagenase
digestion was able to produce high numbers of viable cells
[0190] Two placentas were obtained, each from a different donor,
and processed according to the procedure detailed in Example 1
through Example 2, except a collagenase II concentration of 244
U/mL, as described above. A cell count was performed immediately
following digestion to determine the number of viable cells per
gram of tissue that each enzyme was able to digest away off the
tissue. The data are presented in FIG. 2.
[0191] The placentas were further processed as described in Example
3 through Example 6. Before freezing and after thawing, cells were
counted using a hemocytometer and trypan blue staining was used to
distinguish live cells. The data are presented in FIG. 3.
[0192] Surprisingly, a substantial population of cells was isolated
by digestion of less than 1 hr (e.g. 10 min). Digesting the tissue
for only 10 min allowed the loosening and removal cells from the
tissue without completely breaking up the tissue. In this manner,
it was possible to separate the collagenase II/cell mixture from
the chorionic membrane. The inventors discovered that 10 min was an
adequate amount of digestion time and allowed for variances
introduced as a result of donor variability. The digestion process
allows isolation of as many live cells as possible while not
disrupting the tissue integrity of the chorion membrane to a degree
that makes it impossible to manipulate further. The chorion
membrane could then be disrupted to produce a placental dispersion
that was rich in placental factors while the cells could be
isolated from the enzyme solution and then reintroduced to the
dispersion to form the placental product.
Example 8
Isolation of Cells without Complete Digestion of Placental
Tissue
[0193] Multiple placental products were prepared and cell counts
were taken immediately following digestion (FIG. 4) and before
freezing and after thawing (FIG. 1), using the procedure described
in Example 7. Cells were counted using a hemocytometer and trypan
blue staining was used to distinguish live cells. All cell count
data was pooled and a mean was calculated.
[0194] As depicted in FIG. 4, digestion of an intact membrane as
taught herein produces a surprising number of cells, and does so
without mechanical disruption of the membrane. Also depicted in
FIG. 4, digestion of a membrane as taught herein produces a
surprisingly high ratio of viable to non-viable cells.
[0195] As depicted in FIG. 1, a fresh placental product of the
present invention comprises surprisingly high cell viability. Also
as depicted in FIG. 1, a placental product of the present invention
subjected to a freeze-thaw cycle comprises surprisingly high cell
viability. Also as depicted in FIG. 1, a placental product of the
present invention, cell viability is retained surprisingly well
after a freeze-thaw cycle.
Example 9
Isolation of Placental Cells
[0196] Manufacturing methods were explored to obtain superior
recovery of live cells in the placental dispersion. Specifically,
an experiment was performed to determine the level of viable cells
in a placental product manufactured with or without a step of cell
isolation before homogenization. Briefly, a placenta prepared
according to the procedure detailed in Example 1. The resulting
chorion membrane was then divided into equal halves. Half the
tissue was processed as described in Example 2 through Example 5
and the other half was processed in the same manner but without
cell isolation (collagenase II digestion) prior to homogenization
followed by recombining the isolated cells with the dispersion.
Cells were counted using a hemocytometer and trypan blue staining
was used to distinguish live cells. The data are presented in FIG.
5.
[0197] Results indicate that without prior digestion,
homogenization eliminates virtually all viable cells from the end
dispersion. Surprisingly, a placental product contains a
substantially greater number of viable cells and is provides
enhanced therapeutic efficacy when manufactured with a step of cell
isolation before homogenization.
Example 10
Profile of a Placental Product
[0198] Multiple placental products were prepared, each from a
different donor, according to the procedure detailed in Example 1
through Example 6 and placental factors were analyzed. Briefly, 1
mL of homogenate from each placental product was centrifuged at
14,000 rpm in a microcentrifuge for 10 min.
[0199] The resulting supernatant from each sample was collected as
a test sample. Negative control samples consisted of 5% DMSO in
saline (cryopreservation solution) and positive control samples
consisted of cryopreservation solution with a known concentration
of spiked recombinant proteins (bFGF, EGF, and VEGF). Protein
profiles comprising placental factors listed in Table 1 were
obtained using the SearchLight protein array assay (Aushon
Biosystems). Results are indicated in Table 1 as a minimum and
maximum expression levels (pg/mL) in a pool of four donors. Since
the supernatant is analyzed rather than the complete homogenate, it
is likely that protein level estimates are below actual
concentrations in each chorion membrane homogenate containing live
cells. The levels of VEGF and bFGF in each sample were confirmed by
ELISA.
[0200] Surprisingly, many placental factors were detectable at
levels that are known to be influential for burn wound healing as
well as in the treatment of other indications.
[0201] As seen from the data in Table 1, placental products of the
present invention comprise a therapeutic profile of placental
factors.
[0202] Table 2 sets forth a therapeutic profile of placental
products. Only now, with the teaching herein, the skilled artisan
can examine the placental factors, consider the functional role as
set forth in Table 3, and assess the value of a placental factor in
wound repair.
TABLE-US-00001 TABLE 1 Therapeutic Profile of Factors in the
Placental Products Min. Max. Mean Protein (pg/mL) (pg/mL) (pg/mL)
Function MMP1 2210.07 3468.94 2808.12 Matrix and growth factor
degradation, facilitate cell migration MMP2 8207.46 70964.65
25648.74 MMP3 241.76 615.23 454.49 MMP7 79.78 4429.02 1190.31 MMP8
778.03 4661.35 2821.20 MMP9 32879.10 149579.10 71487.03 MMP10
6728.94 22686.00 14688.40 MMP13 TLTD TLTD TLTD TIMP1 18739.41
315870.30 116341.69 Inhibit activity of MMPs, angiogenic TIMP2
7160.87 60711.15 21335.46 TSP1 TLTD TLTD TLTD Regulate TGF.beta.
activity, anti-angiogenic TSP2 1123.02 18784.67 6190.03 TGF.alpha.
TLTD TLTD TLTD Stimulate growth and migration TGF.beta.1 1041.50
6572.83 2661.65 Promote angiogenesis, also proliferative and
migration stimulatory effects TGF.beta.2 91.81 1809.81 558.53
Promote angiogenesis, also proliferative and migration stimulatory
effects TGF.beta.3 77.02 146.31 104.35 Inhibit scar formation bFGF
(FGF-2) 3554.58 11856.91 7479.40 Promote angiogenesis, also
proliferative and migration stimulatory effects KGF (FGF-7) 14.15
111.58 45.72 Stimulate cell growth and migration EGF 0.42 3.72 1.57
Stimulate cell growth and migration HB-EGF TLTD TLTD TLTD PDGFAA
39.20 173.52 77.46 Promote angiogenesis, also proliferative and
migration stimulatory effects PDGFAB 495.90 495.90 495.90 PDGFBB
7.73 235.85 70.56 VEGF 13.95 211.17 76.73 Promote angiogenesis,
also proliferative and migration stimulatory effects VEGFC 64.77
178.51 118.71 VEGFD 64.73 85.55 77.34 HGF 9180.77 71280.10 27480.10
Inhibit scar formation, stimulate cell growth and migration PEDF
805.18 805.18 805.18 Stimulate growth and migration ANG2 TLTD TLTD
TLTD Stimulate growth and migration IGFBP1 5022.96 1227128.50
322596.69 Regulate IGF and its proliferative effects IGFBP2 564.62
564.62 564.62 IGFBP3 226.20 809.16 603.93 ACRP30 6403.34 33898.70
16229.15 Regulate growth and activity of keratinocytes Fibronectin
2950999.50 90198200.00 24973399.00 ECM, cellular adhesion,
stimulates growth and migration Alpha2mac 280783.30 4653881.00
1554151.49 Inhibit protease activity, coordinate growth factor
bioavailability IL1ra 961.93 10035.52 3568.27 Anti-inflammatory
NGAL 420.82 2908.38 1592.17 Anti-bacterial SDF1b TLTD TLTD TLTD
Recruit cells from circulation to site of tissue damage TLTD = too
low to detect
TABLE-US-00002 TABLE 2 Therapeutic Profile of Factors in the
Chorionic Membrane Max. Mean Protein Min. (pg/mL) (pg/mL) (pg/mL)
MMP1 2882.87 6582.26 4732.56 MMP2 748.82 949.52 849.17 MMP3 TLTD
TLTD TLTD MMP7 4.46 9.07 6.76 MMP8 TLTD TLTD TLTD MMP9 1259.30
2676.23 1967.77 MMP10 79.31 87.51 83.41 MMP13 TLTD TLTD TLTD TIMP1
17419.86 50712.30 34066.08 TIMP2 640.73 779.98 710.36 TGF.alpha.
TLTD TLTD TLTD bFGF (FGF-2) 351.28 375.05 363.17 KGF (FGF-7) 1.53
3.07 2.30 EGF 0.75 0.75 0.75 HB-EGF 15.40 84.49 49.94 PDGFAA 35.25
39.79 37.52 PDGFAB 14.03 14.43 14.23 PDGFBB 1.29 3.99 2.64 VEGF
8.39 125.16 66.78 VEGFC 51.74 123.45 87.60 VEGFD 14.99 20.42 17.70
HGF 29979.57 50392.75 40186.16 PEDF TLTD TLTD TLTD ANG2 TLTD TLTD
TLTD IGFBP1 934.03 1443.63 1188.83 IGFBP2 134.61 135.86 135.24
IGFBP3 4571.51 11970.15 8270.83 LIF TLTD TLTD TLTD GCSF 0.74 1.22
0.98 TPO TLTD TLTD TLTD PIGF TLTD TLTD TLTD ACRP30 225.35 1213.70
719.52 Alpha2mac 8174.44 9968.59 9071.52 IL1ra 525.53 5168.21
2846.87 NGAL 229.72 938.51 584.11 SDF1b TLTD TLTD TLTD TLTD = too
low to detect
TABLE-US-00003 TABLE 3 Functions of Placental Factors Specific
Proteins Selected Functions Matrix Metalloproteinase 1 (MMP1),
MMP2, 3, 7, 8, 9, 10, 13 Matrix and growth factor degradation,
facilitate cell migration Tissue Inhibitors of MMPs (TIMP1 and
TIMP2) Inhibit activity of MMPs, angiogenic Angiotensin-2 (Ang-2),
Heparin-Bound Epidermal Growth Stimulate growth and migration
Factor (HB-EGF), EGF, FGF-7 (also known as Keratinocyte Growth
Factor-KGF), Placenta Growth Factor (PIGF), Pigment Epithelium
Derived Factor (PEDF), Thrombopoietin (TPO), Transforming Growth
Factor-.alpha. (TGF-.alpha.) Basic Fibroblast Growth Factor basic
(bFGF), Platelet Derived Promote angiogenesis, also Growth Factors
(PDGF) AA, AB and BB, Vascular Endothelial proliferative and
migration stimulatory Growth Factor (VEGF), VEGF-C and VEGF-D
effects TGF-.beta.3, Hepatocyte Growth Factor (HGF) Inhibit scar
formation .alpha.2-macroglobulin Inhibit protease activity,
coordinate growth factor bioavailability Adiponectin (Acrp-30)
Regulate growth and activity of keratinocytes Granulocyte
Colony-Stimulating Factor (G-CSF) Stimulate stem cell migration and
proliferation Interleukin 1 Receptor Antagonist (IL-1RA)
Anti-inflammatory Neutrophil Gelatinase-Associated Lipocalin
(N-GAL) Anti-bacterial Leukemia Inhibitory Factor (LIF) Support of
angiogenic growth factors SDF-1.beta. Recruit cells from
circulation to site of tissue damage Insulin-like Growth Factor
Binding Protein (IGFBP1, 2, 3) Regulate IGF and its proliferative
effects
Example 11
Cell Phenotype
[0203] FACS was performed to determine cell phenotype in a
placental product of the present invention. Placental products were
prepared according to the procedure detailed in Example 1 through
Example 6. The products were thawed and subsequently 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).
[0204] Once the single cell suspensions were prepared, 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 4
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. Results indicate that a placental product derived
from chorion contains live cells which stain positive for MSC
markers (FIG. 6), implicating the presence of MSC-like cells.
TABLE-US-00004 TABLE 4 FACS Antibodies Volume of Cell antibody Cell
marker antibody solution marker Cell marker and label type Cat No.
used type specificity IgG1 isotype-PE BD 559320 5 .mu.L Cell
Isotype control surface CD105-PE Caltag 20 .mu.L Cell MSC marker
MHCD10504 surface CD166-PE BD 559263 80 .mu.L Cell MSC marker
surface CD45-PE BD 555483 10 .mu.L Cell Hematopoetic surface cell
marker
Example 12
Optimization of Cryoprotectants
[0205] A placenta was processed according to the procedure detailed
in Example 1 through Example 2. The resulting digestion suspension
comprising cells was divided into several aliquots, and each
processed according to the procedure detailed in Example 3 through
Example 5 using a different cryoprotectant. Three different
cryoprotectants were analyzed for their ability to enhance the
number of viable cells recovered after freezing and to preserve
protein recovery after freezing. The following cryoprotectant
solutions were tested: [0206] 1. 10% DMSO and 5% HSA in Plasma-Lyte
A (CTR solution) [0207] 2. 5% DMSO and 5% HSA in Plasma-Lyte A
[0208] 3. 10% DMSO in Saline [0209] 4. 5% DMSO in Saline [0210] 5.
10% Glycerol in Saline
[0211] Before freezing and after thawing, cells were counted using
a hemocytometer and trypan blue staining was used to distinguish
live cells. The following formula was used to calculate the number
of cells per mL of homogenate: Cells per ml=(# Cells counted per
four 0.0001 mL squares).times.10,000.times.dilution factor. The
results are depicted in FIG. 7.
Example 13
Time Course Optimization of Collagenase Digestion of Chorionic
Tissue
[0212] To determine the optimal time to digest a placental tissue
such as chorionic tissues in collagenase II, chorionic tissues from
three different donors were analyzed. The tissues were incubated
overnight in an antibiotic cocktail. Each chorionic membrane tissue
was then washed twice to remove antibiotic solution and split into
three pieces. Each piece of tissue was weighed to obtain an initial
weight (0 min.) before being digested for 10, 20, or 30 minutes in
collagenase II solution (300 U/mL).
[0213] At the end of each digestion period, the remaining tissue
was separated from the collagenase II solution containing the
isolated cells by filtering through a 100 um pore cell filter. The
separated tissue was then weighed while the collagenase II solution
containing digested cells was centrifuged. The resulting cell
pellet was resuspended in PBS and counted using a hemocytometer
with trypan blue exclusion.
[0214] The weight of each remaining tissue piece, including the
weight of tissue remaining on the cell filter, was used to
calculate the percent of weight lost by digestion with collagenase
II.
[0215] As shown in FIG. 8, after 10 min. of digestion, about 10% of
the original tissue weight was reduced. Further incubation resulted
in a more dramatic loss of weight. By 30 minutes, nearly half of
the original weight was lost. It was further noted that tissue
digested for longer than 10 min. became extremely difficult to
separate from the collagenase II solution.
[0216] FIG. 8 also shows the number of cells released by
collagenase digestion. After 10 minutes of incubation, a
substantial number of cells were released. However, by 20 minutes,
the number of cells released increased by about 4-fold.
[0217] These results surprising demonstrate that by performing only
a limited collagenase digestion (e.g. about 10 minutes), a
substantial number of placental cells can be released and the
integrity of the placental tissue is maintained. Accordingly, when
the limited collagenase digested placental tissue is subsequently
disrupted, the dispersion retains a substantial amount of its
native character. For example, the inventors generally observe that
after prolonged collagenase digestion (e.g. 30 minutes), the
placental tissue can be passed through a 100 micron filter. This is
in contrast to the limited digestion where substantially less (e.g.
one half or one quarter or less) of the tissue can be passed
through a 100 micron filter.
[0218] When this dispersion is combined with the released placental
cells, a superior therapeutic product is produced.
[0219] In data not shown, no significant change in the viability of
the collagenase-released cells was observed through 30 min. of
digestion.
Example 14
Time Course Optimization of Collagenase Digestion of Amniotic
Tissue
[0220] The limited digestion method of Example 13 was tested for
applicability when the placental tissue is amniotic tissue. The
following procedure was performed: [0221] 1. Process placenta.
[0222] a. Remove amniotic tissue and wash twice in PBS. [0223] b.
Soak amniotic tissue to loosen red blood cells. [0224] i. If
needed, clear red blood cells from tissue using fingers. [0225] c.
Incubate amniotic tissue for 24 hrs. in antibiotic cocktail. [0226]
2. Remove amniotic tissue from antibiotic cocktail and wash twice
in PBS. [0227] 3. Incubate amniotic tissue for 30 min at 37.degree.
C. in 200 mL trypsin solution (0.25%). [0228] 4. Remove amniotic
tissue from trypsin solution and wash twice in PBS. [0229] 5.
Incubate amniotic tissue for 10 min at 37.degree. C. in 200 mL
collagenase II solution (300 U/mL in DMEM). [0230] 6. Remove
amniotic tissue from collagenase II solution and wash twice in PBS.
[0231] 7. Processing of collagenase II and trypsin live cell
suspensions. [0232] a. Centrifuge each suspension at 2000 rcf for 5
min. [0233] b. Pour off each supernatant and replace with 10 mL
PBS. [0234] i. Resuspend cells in PBS to wash. [0235] c. Centrifuge
cell suspension at 2000 rcf for 5 min. [0236] d. Pour off
supernatants and resuspend cells in 2 mL cryprotectant (5% DMSO in
saline). [0237] e. Combine pellets. [0238] 8. Processing of
amniotic tissue. [0239] a. Place amniotic tissue in homogenization
container with a volume of cryoprotectant (mL) equal to the weight
of the amniotic membrane (g). For example, if the amniotic membrane
weight 25 g place it in the homogenization container with 25 mL of
cryoprotectant. [0240] b. Allow the amniotic tissue and
cryoprotectant to sit on ice for at least 10 min. [0241] c.
Homogenize at high speed twice for 5 sec. using a tissue
homogenizer. [0242] 9. Combine isolated live cells with homogenate
and mix thoroughly (the "placental product"). [0243] 10. Aliquot
into vials and place at 4.degree. C. for 30-60 min. [0244] Freeze
at -80.degree. C. until use.
[0245] To determine the mean number of live cells in the amnion
homogenate, multiple placentas were prepared. Each amnion was
processed in one piece, and cell counts were obtained post thaw
after cryopreservation (incubation at 4.degree. C. and subsequent
freezing at -80.degree. C.). All cell count data were pooled, and a
mean was calculated.
[0246] Samples from each donor were also prepared for protein array
analysis. Briefly, 1 mL of homogenate from each donor was
centrifuged at 14,000 rpm in a microcentrifuge for 10 min. The
resulting supernatant from each sample was collected. Supernatants
along with positive and negative controls were sent to Aushon
Biosystems for analysis using their SearchLight protein array
assay. This assay measures the levels of 37 proteins of interest in
each sample. For this experiment, negative control samples
consisted of 5% DMSO in saline (cryopreservation solution), and
positive control samples consisted of cryopreservation solution
with known concentrations of spiked recombinant proteins (bFGF,
EGF, and VEGF).
[0247] FACS analysis of single cell suspensions from the placental
product was performed for the markers CD45, CD 105, and CD 166.
[0248] Results.
[0249] As shown in FIG. 9, limited collagenase digestion of
amniotic membrane tissue resulted in release of a substantial
number of live placental cells.
[0250] As shown in Table 5, limited collagenase digestion of
amniotic membrane tissue preserved placental factors in the
placental dispersion made therefrom.
[0251] When Example 13 and Example 14 are considered together, it
is now concluded that limited collagenase digestion of placental
tissue, whether it be chorion tissue, amniotic tissue, or other
tissue of placental origin, results unexpectedly in:
[0252] Substantial numbers of release live placental cells;
[0253] Preserved endogenous placental factors;
[0254] Preserved endogenous placental protein (e.g. matrix
proteins); and
[0255] A therapeutically effective product.
TABLE-US-00005 TABLE 5 Therapeutic Profiles of Amnion-Derived
Placental Products Max. Mean Protein Min. (pg/mL) (pg/mL) (pg/mL)
MMP1 6697.73 10010.27 8354 MMP2 5456.52 53432.45 29444.49 MMP3
570.97 579.1 575.04 MMP7 74.11 207.31 140.71 MMP8 3829.63 3978.42
3904.03 MMP9 11735.19 43661.63 27698.41 MMP10 38916.81 51526.9
45221.86 MMP13 TLTD TLTD TLTD TIMP1 31427.94 78147 54787.47 TIMP2
6149.25 23167.29 14658.27 TSP1 TLTD TLTD TLTD TSP2 7741.98 13312.64
10527.31 TGF.alpha. TLTD TLTD TLTD TGF.beta.1 85.17 350.51 217.84
TGF.beta.2 47.98 58.6 53.29 bFGF (FGF-2) 19305.72 23427.48 21366.6
KGF (FGF-7) 70.39 94.29 82.34 EGF 13.71 69.55 41.63 HB-EGF TLTD
TLTD TLTD PDGFAA 14.47 27.93 21.2 PDGFAB TLTD TLTD TLTD PDGFBB 7.49
12.34 9.91 VEGF 346.3 1058.85 702.57 VEGFC 114.35 220.27 167.31
VEGFD 49.54 75.29 62.42 HGF 12068.53 17408.53 14738.53 PEDF TLTD
TLTD TLTD ANG2 TLTD TLTD TLTD IGFBP1 128.6 159.84 144.22 IGFBP2
TLTD TLTD TLTD IGFBP3 699.01 1349.06 1024.04 ACRP30 6677.35
11232.13 8954.74 Fibronectin 141595.2 254184.05 197889.63 Alpha2mac
421402.95 790851 606126.98 IL1ra 7542.74 10535.55 9039.14 NGAL
1521.63 3283.59 2402.61 SDF1b TLTD TLTD TLTD TLTD = too low to
detect
Example 15
Live Cells from the Placental Dispersions and the Placental Cell
Components of the Placental Product
[0256] The manufacturing steps taught here (e.g. limited
collagenase digestion, removal of placental cells before placental
tissue disruption, and limited disruption methods) result in a
highly effective therapeutic product. The relative role of the
placental dispersion and the placental cells components were
evaluated for respective role in providing live cells.
[0257] Chorionic tissue was obtained from placental tissue of 9
subjects and the placental cells (e.g. collagenase-released) and
placental dispersion was assessed for the number of live cells.
TABLE-US-00006 TABLE 6 Placental Cells from Placental Cell and
Placental Dispersion Fractions Cells in the Placental Cells in
Theoretical cell fraction the Placental cells in the Donor
(collagenase-released) dispersion fraction placental product D144
3.84E+05 7.95E+06 8.33E+06 D145 8.40E+05 1.25E+07 1.33E+07 D146
1.60E+05 7.84E+06 8.00E+06 D147 2.17E+07 5.70E+06 2.74E+07 D153
3.26E+06 1.64E+07 1.97E+07 D154 3.70E+05 1.07E+07 1.11E+07 D155
2.08E+06 7.10E+06 9.18E+06 D156 4.90E+05 1.26E+07 1.31E+07 Mean
3.66E+06 1.01E+07 1.38E+07
[0258] As shown in Table 6, 21% to 98% of the cells in the
placental products were derived from the placental dispersion
component. Thus, the methods of the present invention unexpectedly
preserve important placental factors and live cells in the
placental dispersion and also provide substantial numbers of live
cells from the placental cell (collagenase-released) component.
Example 16
Hypoxia Treatment
[0259] Results from private studies indicate that hypoxia induces
many proteins having beneficial functions in the process of burn
wound healing. However, the extent to which hypoxia effects cell
growth and protein expression depends on the specific conditions of
its application. Therefore, several experiments were performed to
determine if hypoxia could enhance the effectiveness of
chorion-derived placental products.
[0260] A placenta was processed according to the procedure detailed
in Example 1, except the chorionic membrane was divided into two
halves before treatment with the antibiotic cocktail. One half of
the chorionic membrane tissue was incubated under hypoxic
conditions (1% O2) while the other was incubated under normal cell
culture conditions (.about.20% O2). Each half was then process as
described in Example 2 through Example 5. Before freezing and after
thawing, cells were counted using a hemocytometer and trypan blue
staining was used to distinguish live cells. The results are
depicted in FIG. 10.
Example 17
Hypoxia Treatment and Cryoprotectants
[0261] A placenta was processed according to the procedure detailed
in Example 15 except that the digests from each half of the
chorionic membrane were further split and formulated with different
cryoprotectants, as in Example 12. Before freezing and after
thawing, cells were counted using a hemocytometer and trypan blue
staining was used to distinguish live cells. The data are presented
in FIG. 11. As depicted in FIG. 11, processing under normoxic
conditions provides superior cell viability. Also as depicted in
FIG. 11, subjecting the chorion to hypoxic conditions may be
detrimental to cell viability.
Example 18
Growth Factors are Expressed for a Minimum of 14 days
[0262] 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
placental product derived from the chorionic 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.
[0263] Placental product derived from the chorionic membrane 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 product was collected and centrifuged at
16,000 g for 10 min to collect the supernatant. The supernatants
were then run on ELISAs for bFGF and VEGF. FIG. 12 illustrates the
duration of two key wound healing proteins, bFGF and VEGF, at 3, 7
and 14 days. Although the expression of bFGF goes down with time,
it should be noted that significant levels of bFGF was present even
out to 14 days. Interestingly, the expression of VEGF increased
with time, which could be due to continued active expression of
VEGF from the viable cells within the placental product derived
from the chorionic membrane.
Example 19
Interferon 2.alpha. (IFN-2.alpha.) and Transforming Growth
Factor-.beta.3 (TGF-.beta.3)
[0264] Interferon-2.alpha. and TGF-.beta.3 have been described in
the literature as playing critical roles in the prevention of scar
and contracture formation (Kwan et al., Hand Clin, 2009, 25:511;
Tredget et al., Surg Clin North Am 1997, 77:701). IFN-2.alpha. is
known to decrease collagen and fibronectin synthesis and
fibroblast-mediated wound contracture. Clinically, IFN-2.alpha. has
been administered subcutaneously and shown to improve scar quality
(Nedelec et al, Lab Clin Med 1995, 126:474). TGF-.beta.3 regulates
the deposition of extracellular matrix and has been shown to
decrease scar formation when injected in rodent cutaneous wound
models. Clinically, TGF-.beta.3 has been shown to improve scar
appearance when injected at the wound site (Occleston et al., J
Biomater Sci Polym Ed 2008, 19:1047). Placental product derived
from the chorionic membrane described in this invention has been
analyzed for the presence of IFN-2.alpha. and TGF-.beta.3. Briefly,
placental product derived from the chorionic membrane was thawed
and centrifuged at 16,000 g to collect supernatants. Supernatants
were analyzed on a commercially available ELISA kit from MabTech
(IFN-2.alpha.) and R&D Systems (TGF-.beta.3). FIG. 13 shows
significant expression of IFN-2.alpha. and TGF-.beta.3 in placenta
products derived from the chorionic membrane.
Example 20
Tissue Reparative Proteins in Chorionic Membrane Homogenates
[0265] Placental product derived from the chorionic membrane was
analyzed for the presence of proteins that are important in tissue
repair.
[0266] Placental products derived from chorionic membranes
described in this invention were analyzed for the presence of these
tissue reparative proteins. Briefly, placental product derived from
the chorionic membrane was incubated at 37.degree. C..+-.2.degree.
C. for 72 hrs. The product was centrifuged, and the supernatant was
analyzed on commercially available ELISA kits from R&D Systems.
FIG. 14 shows significant expression of BMP-2, BMP-4, BMP-7, PLAB,
PIGF, and IGF-1 in several donors of placental products derived
from chorionic membranes.
[0267] 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.
[0268] 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 21
Differentiation Capacity of Cells Derived from the Chorionic
Membrane
[0269] Placental 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.
[0270] The expression of specific cellular markers has already been
described in Example 20. 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.
[0271] FIG. 15-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.
15-B.
[0272] 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.
[0273] FIG. 15-C shows a representative image of passage 2 cells
isolated and expanded from placental product derived from the
chorionic membrane staining positively for alkaline
phosphatase.
* * * * *