DISPERSIONS

RELATED APPLICATIONS

001 This application claims priority to:

002 U.S. Provisional Applications Ser. No, 61/338,464 entitled "Selectively Immunodepleted Chorionic Membranes" , filed on February 18, 2010 bearing Docket No. 22924US01 ,

003 U.S. Provisional Applications Ser. No, 61/338,489 entitled "Selectively Immunodepleted Amniotic Membranes" , filed on February 18, 2010 bearing Docket No. 22925US01 , and

004 U.S. Provisional Applications Ser. No, 61/369,562 entitled "Therapeutic Products Comprising Vitalized Placental Dispersions filed on July 30, 2010 bearing Docket No 23498US01 , the contents of which are hereby incorporated by reference in their entireties.

005 This application is being co-filed on February 18, 201 1 with, and incorporates by reference, applications entitled:

006 "Immunocompatible Chorionic Membrane Products",

007 "Methods of Manufacture of Immunocompatible Chorionic Membrane Products",

008 "Immunocompatible Amniotic Membrane Products",

009 "Methods of Manufacture of Immunocompatible Amniotic Membrane Products", and

0010 "Methods of Manufacture of Therapeutic Products Comprising Vitalized Placental Dispersions". TECHNICAL FIELD

001 1 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 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 making placental products.

0018 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.

0019 Optionally, the placental dispersion is a homogenate.

0020 Optionally, placental factors present include extracellular matrix components.

0021 Optionally, the placental dispersion comprises one or more placental factors set forth in Table 1 , Table 2, Table 3, or Table 5.

0022 Optionally, the placental cells comprise stromal cells such as MSCs (mesenchymal stem cells) and PSCs (placental stem cells).

0023 In one embodiment, products of the present invention are produced by a parallel processing method that comprises:

i) obtaining a first placental (e.g. amniotic or chorionic) tissue; ii) obtaining placental cells from the first placental tissue;

iii) obtaining a second placental (e.g. amniotic or chorionic) tissue;

iv) disrupting the second placental tissue to form a dispersion comprising

placental factors;

v) combining the placental cells and the dispersion to form the placental product.

0024 Optionally, the first placental tissue and the second placental tissue are autologous to each other, for example, derived from the same donor.

0025 In one embodiment, products of the present invention are produced by 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.

0026 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).

0027 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.

0028 In another embodiment, the method of making a placental product comprises:

i) obtaining a placental (e.g. amniotic or chorionic) tissue;

ii) exposing the placental tissue to collagenase;

iii) dividing the placental tissue into a first portion and a second portion;

iv) isolating placental cells from the first placental portion;

v) disrupting the second placental portion to form a dispersion comprising

placental factors; and vi) combining the placental cells and the placental dispersion to form the placental product.

vii) In another embodiment, the method of making a placental product comprises: viii) obtaining a placental (e.g. amniotic or chorionic) tissue;

ix) exposing the placental tissue to a collagenase for a time sufficient to release placental cells;

x) isolating the released placental cells from the collagenase exposed placental tissue;

xi) disrupting the collagenase exposed placental tissue to form a dispersion

comprising placental factors; and

xii) combining the placental cells and the placental dispersion to form the

placental product.

BRIEF DESCRIPTION OF THE DRAWINGS

0029 Figure 1 depicts cell viability, before and after a freeze-thaw cycle of a placental product comprising isolated cells and a placental dispersion.

0030 Figure 2 depicts recovery of viable cells isolated by digestion.

0031 Figure 3 depicts cell viability, before and after a freeze-thaw cycle of a placental product comprising isolated cells and a placental dispersion.

0032 Figure 4 depicts recovery of viable cells isolated by digestion.

0033 Figure 5 depicts the level of viable cells in a placental product made with or without a step of cell isolation before tissue disruption.

0034 Figure 6 depicts cell phenotype of cells in a placental product.

0035 Figure 7 depicts cell viability using various cryoprotectants

0036 Figure 8 depicts placental tissue weight and live cells recovered following collagenase treatment of various incubation times.

0037 Figure 9 depicts the number of collagenase-released cells from multiple donors. 0038 Figure 10 depicts viable cell level in a placental product when a placenta is subjected to hypoxic or normoxic conditions.

0039 Figure 1 1 depicts cell viability when a placenta is subjected to hypoxic or normoxic conditions.

0040 Figure 12 depicts expression of bFGF in placental products for 14 days in culture.

0041 Figure 13 depicts expression of IFN-2a and TGF-B3 in placental products,

0042 Figure 14 BMP-2, BMP-4, BMP-7, PLAB, PIGF, and IGF-1 were detected in placental products derived from the chorionic membrane

0043 Figure 15 . Representative image of passage 2 cells isolated and expanded from a placental product derived from the chorionic membrane

0044 Figure 16 depicts recovery of viable cells isolated by digestion using various collagenase II enzymes.

0045 Figure 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

0046 As used here, the following definitions and abbreviations apply.

0047 "Chorionic tissue" or "Chorionic membrane" means the chorion or a portion thereof, e.g. the trophoblast, the somatic mesoderm, or combinations thereof.

0048 "Examplary" (or "e.g." or "by example") means a non-limiting example.

0049 "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.

0050 "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. 0051 "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.

0052 "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.

0053 "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.

0054 "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, cryosurgery, heat and electrical ablations), and surgical incisions.

Placental Product

Overview

0055 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.

0056 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 ² , about 10 cm ² , about 25cm ² , about 50cm ² , or about 100 cm 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.

0057 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.

0058 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.

Placental Cells

0059 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.

0060 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").

0061 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, III and IV), matrix metalloprotease, neutral proteases, papains, deoxyribonucleases, serine protease (e.g. trypsin, chymotrypsin, elastase), or any combination thereof.

0062 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°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.

0063 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.

0064 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.

Placental Dispersion

0065 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.

0066 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. 0067 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.

0068 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.

Placental Factors

0069 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.

0070 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".

0071 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

0072 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. 0073 The present placental products can optionally comprise a therapeutic profile of one or more of a PDGF (e.g. PDGF-bb), EGF, FGF, TGF-βΙ , TGF- β3, and VEGF and/or one or more of IL-8, IL-6, and MCP-1 .

0074 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.

0075 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, TGFB1 , bFGF, and MMPs (e.g. MMP1 ,2,4,7,8,9, and 10).

0076 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).

0077 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-βΙ . Optionally the concentration of such placental factors is about 25% of the minimum values set forth in in Table 1 and optionally no more than 400% of the maximum concentration set forth in Table 1

0078 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,

0079 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-βΙ and/or TGF-P2), 0080 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.

0081 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

1 1 ,000 pg/mL, as detailed in Example 10. Optionally such FGF-comprising placental products are useful for burn wound healing.

0082 Placental products of the present invention can comprise a therapeutic profile of TGF-βΙ and TGF- 2. An examplary placental product, as detailed in Example 10, comprises bFGF, TGF-βΙ and TGF- 2. Optionally, such placental products are useful when the skin pathology being treated involves an inflammatory or a scaring pathology.

0083 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.

0084 In one embodiment, a placental product (e.g. derived from chorion) comprises one or more protease inhibitors.

0085 In one embodiment, a placental product (e.g. derived from chorion) comprises one or more protease inhibitors and extracellular matrix proteins

0086 In one embodiment, a placental product (e.g. derived from chorion) comprises one or more protease inhibitors and viable cells.

0087 In one embodiment, a placental product (e.g. derived from chorion) comprises one or more protease inhibitors, extracellular matrix proteins, and viable cells.

0088 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

0089 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.

0090 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.

0091 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.

0092 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.

0093 The placental products of the present invention can optionally include one or more antibiotic, emollient, keratolytics agent, humectants, anti-oxidants, preservative, or combinations thereof.

0094 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.

0095 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

Overview

0096 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.

0097 According to the present invention, the placenta is processed to produce the placental dispersion and the placental cells.

0098 In one embodiment, the placental dispersion and the placental cells are derived from a different placenta or different placental portion (e.g. parallel processing). 0099 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).

00100 Manufacturing Method 1 . In one embodiment, a placental product is manufactured by:

obtaining a placental (e.g. chorionic) tissue;

digesting the placental tissue with one or more matrix degrading enzymes (e.g. a collagenase, optionally collagenase II);

obtaining placental cells from the digested placental tissue;

disrupting the digested placental tissue with a tissue disruptor to form a placental dispersion comprising placental factors; and

combining the placental cells and the placental dispersion to form the placental product.

00101 Optional Manufacturing Method 2 In one embodiment, a placental product is manufactured by:

obtaining a first placental (e.g. chorionic) tissue;

digesting the first placental tissue with one or more matrix degrading enzymes (e.g. a collagenase, optionally collagenase II);

obtaining placental cells from the digested first placental tissue;

obtaining a second placental tissue;

disrupting the second placental tissue with a tissue disruptor to form a placental dispersion comprising placental factors; and

combining the placental cells and the placental dispersion to form the placental product.

00102 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. 00103 Examplary placental products of the present invention can be

manufactured or provided with a bandage or wound dressing.

Trophoblast Removal

00104 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:

a. is substantially non-immunogenic;

b. provides remarkable healing time; and

c. provides enhanced therapeutic efficacy.

00105 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.

00106 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.

00107 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").

Preservation 00108 A placental product of the present invention may be used fresh or may be preserved for a period of time.

00109 Also as depicted in Figure 1 , a placental product of the present invention, cell viability is retained surprisingly well after a freeze-thaw cycle

001 10 In one embodiment, a placental product is cryopreserved. A placental product may be cryopreserved by freezing (e.g. a -80° 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°C for 30-60 min, and then incubating at -80°C until use. The placental product may then be thawed for use.

001 1 1 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%).

001 12 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.

001 13 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

001 14 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.

001 15 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. 001 16 Placental products can be administered autologously, allogeneically or xenogeneically.

001 17 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.

001 18 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.

001 19 In one embodiment, the injury is an ulcer, for example, a diabetic ulcer (e.g. foot ulcer).

00120 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.

00121 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.).

00122 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).

00123 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.

00124 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.

00125 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.

00126 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 Mar;19(3):290-6), Chen et al.

("Enveloping of periosteum on the hamstring tendon graft in anterior cruciate ligament reconstruction"; Arthroscopy. 2002 May-Jun;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.

00127 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.

00128 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.

00129 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.

00130 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.

00131 In one embodiment, a placental product of the present invention is used to reduce fibrosis by applying the placental product to a wound site.

00132 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).

00133 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. 00134 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.

00135 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.

00136 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. 00137 The citations provided herein are hereby incorporated by reference for the cited subject matter.

00138 In the present specification, use of the singular includes the plural except where specifically indicated.

EXAMPLES

Example 1 Obtaining Placental Tissue

00139 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.

00140 The chorionic membrane was then soaked in an anticoagulant (ACD-A) solution to remove blood clots and then washed again in PBS.

00141 The chorionic membrane was then digested by incubation with dispase II for 30 min. at 37°C. The trophoblast layer was mechanically removed by scraping with fingers and the chorion was washed again in PBS.

00142 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

00143 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°C. The chorionic membrane was then removed, leaving a digestion suspension containing collagenase and placental cells.

00144 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°C cell culture incubator.

Example 3 Obtaining Placental Cells

00145 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.

00146 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

00147 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.

00148 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).

00149 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

00150 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

00151 A placental product (obtained from Example 5) was aliquoted into vials and incubated at 4°C for 30-60 min. The vials were then frozen at -80°C until use. Example 7 Isolation of Cells without Complete Digestion of Placenta

00152 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

00153 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 Figure 2.

00154 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 Figure 3.

00155 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 ll/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 00156 Multiple placental products were prepared and cell counts were taken immediately following digestion (Figure 4) and before freezing and after thawing (Figure 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.

00157 As depicted in Figure 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 Figure 4, digestion of a membrane as taught herein produces a surprisingly high ratio of viable to non-viable cells.

00158 As depicted in Figure 1 , a fresh placental product of the present invention comprises surprisingly high cell viability. Also as depicted in Figure 1 , a placental product of the present invention subjected to a freeze-thaw cycle comprises surprisingly high cell viability. Also as depicted in Figure 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

00159 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 Figure 5.

00160 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

00161 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.

00162 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 inTable 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.

00163 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.

00164 As seen from the data in Table 1 , placental products of the present invention comprise a therapeutic profile of placental factors.

00165 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.

00166

Table 1 Therapeutic Profile of Factors in the Placental Products

Min. Max. Mean

Protein (pq/mL) (pq/mL) (pq/mL) Function

Matrix and growth factor degradation, facilitate cell

M P1 2210.07 3468.94 2808.12 migration

MMP2 8207.46 70964.65 25648.74

M P3 241.76 615.23 454.49

M P7 79.78 4429.02 1190.31

MM PS 778.03 4661.35 2821.20

M P9 32879.10 149579.10 71487.03

P10 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 TGFp activity, anti-angiogenic

TSP2 1123.02 18784.67 6190.03

TGFrx TLTD TLTD TLTD Stimulate growth and migration

Promote angiogenesis, also proliferative and migration TGFB1 1041.50 6572.83 2661.65 stimulatory effects

Promote angiogenesis, also proliferative and migration TGFfs2 91.81 1809.81 558.53 stimulatory effects

IGF 33 77.02 146.31 104.35 Inhibit scar formation

Promote angiogenesis, also proliferative and migration bFGF (FGF-2) 3554.58 11856.91 7479.40 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

Promote angiogenesis, also proliferative and migration

VEGF 13.95 211.17 76.73 stimulatory effects

VEGFC 64.77 178.51 118.71

VEGFD 64.73 85.55 77.34

Inhibit scar formation, stimulate cell growth and

HGF 9180.77 71280.10 27480.10 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

ECM, cellular adhesion, stimulates growth and

Fibronectin 2950999.50 90198200.00 24973399.00 migration

Inhibit protease activity, coordinate growth factor

Aipha2mac 280783.30 4653881.00 1554151.49 bioavailability

iL1 ra 961.93 10035.52 3568.27 Anti-inflammatory

GAL 420.82 2908.38 1592.17 Anti-bacterial

SDF1 b TLTD TLTD TLTD Recruit cells from circulation to site of tissue damage

TLTD - too low to detect

Table 2 Therapeutic Profile of Factors in the Chorionic Membrane

Max. Mean

Protein Min. (pg/mL) (pq/mL) (pq/mL) P1 2882.87 6582.26 4732.56

M P2 748.82 949.52 849.17

P3 TLTD TLTD TLTD P7 4.46 9.07 6.76 MP8 TLTD TLTD TLTD

P9 1259.30 2676.23 1967.77 MP10 79.31 87.51 83.41

M P13 TLTD TLTD TLTD

TIMP1 17419.86 50712.30 34066.08

TI P2 640.73 779.98 710.36

TGFrx TLTD TLTD TLTD bFGF (FGF-2) 351.28 375.05 363.17

GF (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

A G2 TLTD TLTD TLTD

IGFBP1 934.03 1443.63 1188.83

IGFBP2 134.61 135.86 135.24

IGFSP3 4571.51 11970.15 8270.83

L!F TLTD TLTD TLTD

GCSF 0.74 1.22 0.98

TPO TLTD TLTD TLTD

PiGF TLTD TLTD TLTD

AC P30 225.35 1213.70 719.52

Alpha2mac 8174.44 9968.59 9071.52

IL1 ra 525.53 5168.21 2846.87

NGAL 229.72 938.51 584.11

SDF1 b TLTD TLTD TLTD

TLTD - too low to detect Table 3 Functions of Placental Factors

Specific Proteins Selected Functions

Matrix and growth factor degradation,

Matrix Metalloproteinase 1 (MMP1 ), MMP2, 3, 7, 8, 9, 10, 13

facilitate cell migration

Tissue Inhibitors of MMPs (TIMP1 and TIMP2) Inhibit activity of MMPs, angiogenic

Angiotensin-2 (Ang-2), Heparin-Bound Epidermal Growth

Factor (HB-EGF), EGF, FGF-7 (also known as Keratinocyte

Growth Factor-KGF), Placenta Growth Factor (PIGF), Pigment Stimulate growth and migration Epithelium Derived Factor (PEDF), Thrombopoietin (TPO),

Transforming Growth Factor-a (TGF-a)

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- 3, Hepatocyte Growth Factor (HGF) Inhibit scar formation

Inhibit protease activity, coordinate a2-macroglobulin

growth factor bioavailability

Regulate growth and activity of

Adiponectin (Acrp-30)

keratinocytes

Stimulate stem cell migration and

Granulocyte Colony-Stimulating Factor (G-CSF)

proliferation

Interleukin 1 Receptor Antagonist (IL-1 RA) Anti-inflammatory

Neutrophil Gelatinase-Associated Lipocalin (N-GAL) Anti-bacterial

Leukemia Inhibitory Factor (LIF) Support of angiogenic growth factors

Recruit cells from circulation to site of

SDF-Ι β

tissue damage

Regulate IGF and its proliferative

Insulin-like Growth Factor Binding Protein (IGFBP1 , 2, 3)

effects Example 11 Cell Phenotype

00167 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 μπΊ 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 ₃ + 1 % FBS).

00168 Once the single cell suspensions were prepared, a minimum of 1 x10 ⁵ cells in 100 μΙ_ 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 μΙ_ 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 (Figure 6), implicating the presence of MSC-like cells.

Table 4 FACS Antibodies

Cell marker antibody Cat No. Volume of Cell Cell marker and label type antibody marker specificity solution type

used

lgG1 isotype-PE BD 559320 5 μΙ_ Cell Isotype control surface

CD105-PE Caltag 20 μΙ_ Cell MSC marker

MHCD10504 surface

CD166-PE BD 559263 80 μΙ_ Cell MSC marker surface

CD45-PE BD 555483 10 μΙ_ Cell Hematopoetic surface cell marker Example 12 Optimization of Cryo protectants

00169 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 cyroprotectant. 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:

1 . 10% DMSO and 5% HSA in Plasma-Lyte A (CTR solution)

2. 5% DMSO and 5% HSA in Plasma-Lyte A

3. 10% DMSO in Saline

4. 5% DMSO in Saline

5. 10% Glycerol in Saline

00170 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) X 10,000 X dilution factor. The results are depicted in Figure 7.

Example 13 Time Course Optimization of Collagenase Digestion of Chorionic Tissue.

00171 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).

00172 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 urn 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.

00173 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.

00174 As shown in Figure 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.

00175 Figure 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.

00176 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.

00177 When this dispersion is combined with the released placental cells, a superior therapeutic product is produced.

00178 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 CoMagenase Digestion of Amniotic Tissue

00179 The limited digestion method of Example 13 was tested for applicability when the placental tissue is amniotic tissue. The following procedure was

performed:

1 . Process placenta.

a. Remove amniotic tissue and wash twice in PBS.

b. Soak amniotic tissue to loosen red blood cells.

i. If needed, clear red blood cells from tissue using fingers.

c. Incubate amniotic tissue for 24 hrs. in antibiotic cocktail.

2. Remove amniotic tissue from antibiotic cocktail and wash twice in PBS.

3. Incubate amniotic tissue for 30 min at 37°C in 200 mL trypsin solution (0.25%).

4. Remove amniotic tissue from trypsin solution and wash twice in PBS.

5. Incubate amniotic tissue for 10 min at 37°C in 200 mL collagenase II solution (300 U/mL in DMEM).

6. Remove amniotic tissue from collagenase II solution and wash twice in PBS.

7. Processing of collagenase II and trypsin live cell suspensions.

a. Centrifuge each suspension at 2000 rcf for 5 min.

b. Pour off each supernatant and replace with 10 mL PBS.

i. Resuspend cells in PBS to wash.

c. Centrifuge cell suspension at 2000 rcf for 5 min.

d. Pour off supernatants and resuspend cells in 2 mL cryprotectant (5% DMSO in saline).

e. Combine pellets.

8. Processing of amniotic tissue. 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. b. Allow the amniotic tissue and cryoprotectant to sit on ice for at least 10 min.

c. Homogenize at high speed twice for 5 sec. using a tissue homogenizer.

9. Combine isolated live cells with homogenate and mix thoroughly (the "placental product").

10. Aliquot into vials and place at 4°C for 30-60 min.

Freeze at -80°C until use.

00180 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°C and subsequent freezing at -80°C). All cell count data were pooled, and a mean was calculated.

00181 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).

00182 FACS analysis of single cell suspensions from the placental product was performed for the markers CD45, CD 105, and CD 166.

00183 Results. 00184 As shown in Figure 9, limited collagenase digestion of amniotic membrane tissue resulted in release of a substantial number of live placental cells.

00185 As shown in Table 5, limited collagenase digestion of amniotic membrane tissue preserved placental factors in the placental dispersion made therefrom.

00186 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:

00187 Substantial numbers of release live placental cells;

00188 Preserved endogenous placental factors;

00189 Preserved endogenous placental protein (e.g. matrix proteins); and

00190 A therapeutically effective product.

Table 5 Therapeutic Profiles of Amnion-Derived Placental Products

Max. Mean

Protein Min. (pg/mL) (pq/mL) (pq/mL)

M P1 6697.73 10010.27 8354

M P2 5456.52 53432.45 29444.49

MMP3 570.97 579.1 575.04

M P7 74.11 207.31 140.71

P8 3829.63 3978.42 3904.03

MMP9 11735.19 43661.63 27698.41

MP10 38916.81 51526.9 45221.86

P13 TLTD TLTD TLTD

T! PI 31427.94 78147 54787.47

TIMP2 6149.25 23167.29 14658.27

TSP1 TLTD TLTD TLTD

TSP2 7741.98 13312.64 10527.31

TGFrx TLTD TLTD TLTD

TGF- i1 85.17 350.51 217.84

TGF[32 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

IL1 ra 7542.74 10535.55 9039.14

NGAL 1521.63 3283.59 2402.61

SDF1 b 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

00191 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.

00192 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 6 Placental Cells from Placental Cell and Placental Dispersion Fractions

Cells in the Placental

cell fraction Cells in the Placental Theoretical 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

00193 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

00194 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.

00195 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 % 02) while the other was incubated under normal cell culture conditions (-20% 02). 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 Figure 10.

Example 17 Hypoxia Treatment and Cryoprotectants

00196 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 Figure 1 1 . As depicted in Figure 1 1 , processing under normoxic conditions provides superior cell viability. Also as depicted in Figure 1 1 , subjecting the chorion to hypoxic conditions may be detrimental to cell viability. Example 18 Growth Factors are Expressed for a Minimum of 14 days

00197 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.

00198 Placental product derived from the chorionic membrane were thawed and plated onto tissue culture wells and incubated at 37°C ± 2°C for 3, 7, and 14 days. At each time point, a sample of the product was collected and centrifuged at 16,000g for 10 min to collect the supernatant. The supernatants were then run on ELISAs for bFGF and VEGF. Figure 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 2a (IFN-2a) and Transforming Growth Factor- 3 (TGF- β3)

00199 lnterferon-2a and TGF-P3 have been described in the literature as playing critical roles in the prevention of scar and contracture formation (Kwan et al., Hand Clin, 2009, 25:51 1 ; Tredget et al., Surg Clin North Am 1997, 77:701 ). IFN-2a is known to decrease collagen and fibronectin synthesis and fibroblast-mediated wound contracture. Clinically, IFN-2a has been administered subcutaneously and shown to improve scar quality (Nedelec et al, Lab Clin Med 1995, 126:474). TGF- β3 regulates the deposition of extracellular matrix and has been shown to decrease scar formation when injected in rodent cutaneous wound models. Clinically, TGF- β3 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-2a and TGF- 3. Briefly, placental product derived from the chorionic membrane was thawed and centrifuged at 16,000g to collect supernatants.

Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2a) and R&D Systems (TGF- 3). Figure 13 shows significant expression of IFN-2a and TGF-P3 in placenta products derived from the chorionic membrane.

Example 20 Tissue Reparative Proteins in Chorionic Membrane Homogenates

00200 Placental product derived from the chorionic membrane was analyzed for the presence of proteins that are important in tissue repair.

00201 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°C ± 2°C for 72 hrs. The product was centrifuged, and the supernatant was analyzed on commercially available ELISA kits from R&D Systems. Figure 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.

00202 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.

00203 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

00204 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.

00205 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°C ± 2°C and expanded.

00206 Figure 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 Figure 15-B.

00207 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'-lndolylphosphate 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.

00208 Figure 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.