KR20170002572A - Immunocompatible amniotic membrane products - Google Patents

Abstract

There is provided herein a placental membrane product comprising an immunocompetent amniotic membrane. Such placental membrane products may be cryopreserved and contain therapeutic agents and viable cells after thawing. Placental membrane products are useful in wound healing and other methods of promoting tissue repair / regeneration and healing, as they promote neovascularization, reduce inflammation, inhibit proteases and free radical oxidation, and reduce scar formation Do. The present technology relates to a product for protecting a wounded or damaged tissue, or a cover for preventing adhesion, which excludes bacteria, inhibits bacterial activity and / or promotes healing or growth of tissue. The field also relates to methods of making and using such membrane-derived products.

Description

IMMUNOCOMPATIBLE AMNIOTIC MEMBRANE PRODUCTS [0002]

Related application

This application is co-filed on May 7, 2014, the full text of which is incorporated by reference as being an invention entitled " Immunocompatible Chorionic Membrane Products ", and Therapeutic Placental Compositions, Methods Of Making And Methods Of Use.

Technical field

The present technology relates to methods and products for facilitating or ameliorating wound healing, including, for example, placental membrane-derived products and cryopreserved products, and at least one of the following: stimulation of neovascularization, growth factor secretion, ≪ / RTI > and free radical oxidation), or a method of promoting healing in the vicinity of or at the wound site. The present technology relates to a product for protecting a wounded or damaged tissue, or a cover for preventing adhesion, which product excludes bacteria, inhibits bacterial activity, or promotes tissue healing or growth. An example of such placental membranes is amniotic membrane. The field also relates to methods of making and using such membrane-derived products.

New or de-saturated placenta membranes have been used topically in surgical applications, at least since 1910, when Johns Hopkins Hospital reported the use of placenta membranes for dermal use. Separate amniotic and chorionic membranes were used to treat subsequent burn or ulcerated surfaces. During the 1950s and 1960s, amniotic membrane boiled in Troensegaard-Hansen was applied to chronic leg ulcers.

Human amniotic membrane (AM) is derived from amniotic membrane and is located at the innermost part of the membrane of the amniotic membrane. It is approximately 0.02 to 0.5 mm thick. AM consists of five layers: a monolayer of epithelial cells remains in the basement membrane and contacts the amniotic fluid. The basal layer of connective tissue attaches to the basement membrane. The connective tissue consists of three structural layers, a dense layer, a fibroblast layer (sometimes referred to as a hypodermic layer), and a sponge layer. The spongy layer is adjacent to the chorion. Amniotic membrane is essentially free of vasculature.

Both new and frozen AM have been used for wound healing remedies. AM contains a number of factors that can contribute to wound healing, such as, for example, extracellular matrix, growth factors, and viable cells. Some preservation methods can maintain some levels of factors such as substrates or growth factors, but preserving viable cell levels provide a challenge. When a new AM is used, the risk of disease transmission is increased. According to published reports, the new amnion tissue shows high cell viability (e.g., greater than 70%), but cell viability is rapidly reduced during storage (e.g., 28 days , To reduce the amount of viable cells in the range of about 15% to 35%. Freezing for a period of time (e.g., 3 weeks) has been shown to reduce cell viability in the range of about 13% to 18% regardless of temperature or medium.

Lee and Tseng report cryopreservation of AM in glycerol and Dulbeccos Modified Eagle medium (DMEM) at 80 ° C, even if such cryopreservation dramatically reduces cell viability . According to an open report, storage of glycerol in AM caused cell death immediately. Glycerol cryopreserved AM (-80 캜) and glycerol-preserved AM (-4 캜) are sufficient to provide a substrate for wound healing, but do not provide sufficient cell viability to confer biological effectiveness.

Gajiwala and Gajiwala report the preservation of AM by freeze-drying (lyophilization) and gamma-radiation sterilization. According to this method, AM is sterilized at 60 ° C, treated with 70% ethanol, and lyophilized to remove most of the remaining moisture. The AM is then sterilized by exposure to 25 kGy gamma-radiation in a cobalt 60 gamma chamber or in an ISO-approved radiation facility. Sterile AM can be stored at room temperature for up to 6 months.

Gomes reports the preservation of AM by sterilization in ethylene oxide followed by lyophilization.

Rama et al reported cryopreservation of AM in 10% dimethyl sulfoxide (DMSO) instead of glycerol, and achieved cell survival of about 40%.

There are two commercially available biologically engineered tissue implants containing viable cells (from neonatal foreskin), Apligraf and Dermagraft (Dermagrain). Both Apples and Derma grafts contain cultured expanded cells. Applescript and dermagraph also do not include detectable levels of certain factors, such as insulin-like growth factor binding protein-1 (IGFBP-1) and adiponectin, which are factors associated with the natural wound healing process. In addition, both the application and the drama graphs may be desirable for wound healing. The specific ratio of substrate metalloproteinase (MMP) to tissue inhibitor (TIMPS) of substrate metalloproteinase (MMP versus TIMP ratio or pronase Versus-protease inhibitor ratio). Because wound healing is a multi-factorial biological process, there are a number of factors needed to properly treat the wound; Lower numbers of viable cells and products with limited density compared to cells present in the skin can increase the number of viable cell populations found in the tissue and lessen wound healing compared to products with an increased number of cell types. Amniotic membrane-derived products that can be used in applications such as wound healing, wound dressing, cosmetic applications, or to promote higher rates of viable cells and, for example, growth factors, antioxidants, anti-inflammatory agents, neovascularization and cytokines Will represent advances in the art to provide as a biological skin substitute comprising a diverse population representing an increased amount of the agent including the agent.

Applescript is a viable bilayer skin substitute made using neonatal perianal keratinocytes and fibroblasts in combination with small type I collagen. As used in this application, Applescript refers to a product available for commercial sale as approved by the FDA in 1998.

Derma grafts are cryopreserved human fibroblasts derived from neoplastic epithelial tissue seeded on synthetic extracellular matrix and bioabsorbable polygalactin mesh scaffolds. According to his product manual, Dermagraft requires three washing steps prior to use, limiting the performance of the Dermagraft as a wound dressing for products requiring less than three washes. As used in this application, Dermagraft refers to a product available for commercial sale as approved by the FDA in 2001.

Engineered wound dressings, such as Apples and Derma grafts, do not provide the greatest potential for wound healing because they contain sub-cellular compositions and thus do not provide adequate wound healing. For example, Applescript and DermaGraft do not contain MSC or native tissue extracellular matrix, so that the ratio between the different factors secreted by the cell does not enable efficient wound healing. In addition, some important factors for wound healing, including EGF, IGFBP1, and adiponectin, are either undetectable or not both Applescript and DermaGraft. Additionally, some factors, including MMP and TIMP, are present at a rate significantly different from that found in the natural wound healing process; This difference, in particular, alters the upstream inflammatory cytokine pathway, which ultimately enables a sub-micron environment at the wound site. In these biotechnically processed products, the substrate composition comprises only type I collagen and may also comprise hyaluronic acid. It differs from the complex structural constituents of the skin, including components such as various collagens (e.g. collagen I, III, IV, V, VI, etc.), elastin, glycoproteins and proteoglycans. The skin also includes intramuscular hepatocyte stem cells lacking in representative examples of biotechnology products, appli grapes, and derma grafts.

Paquet-Fifield et al. Report that hepatic stem cells and fibroblasts are important for wound healing (J Clin Invest, 2009, 119: 2795). Products containing hepatic stem cells and fibroblasts have not yet been described.

Both MMP and TIMP are important factors in wound healing. However, the expression of these proteins must be highly regulated and regulated. Excessive MMP versus TIMP is a marker of poor wound healing (Liu et al, Diabetes Care, 2009, 32: 117; Mwaura et al, Eur J Vasc Endovasc Surg, 2006, 1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).

alpha 2-macroglobulin and / or its receptors are all known as plasma proteins that inactivate proteases from four different mechanistic classes: serine protease, cysteine protease, aspartic protease, and metalloprotease (Borth et al., FASEB J, 1992, 6: 3345; Baker et al., J Cell Sci, 2002, 115: 3719). Other important functions of this protein function as a reservoir of cytokines and growth factors, examples of which include TGF, PDGF and FGF (Asplin et al, Blood, 2001, 97: 3450; Huang et al, J Biol Chem, 1988; 263: 1535). In chronic wounds such as diabetic ulcers or vein ulcers, the presence of high amounts of protease causes rapid degradation of growth factors and delayed wound healing. Thus, placental products containing [alpha] 2-macroglobulin will constitute an advance in the art.

In vitro cell migration assays are important in assessing the potential for wound healing of products. The wound healing process is highly complex and involves a series of structured events controlled by growth factors (Goldman, Adv. Skin Wound Care, 2004, 1:24). These events include increased angiogenesis, infiltration by inflammatory immune cells, and increased cell proliferation. The initiation phase of wound healing deals with the ability of individual cells to become polarized to the wound, to move into the wound area to approach the wound area and reconstitute the surrounding tissue. An analysis that can assess the potential for wound healing of wound treatment by testing the correlation between cell migration and wound healing will provide a margin in the art. As discussed in the disclosure below, aspects of the present technique relate to products and methods that promote neovascularization, promote anti-inflammatory activity, promote antioxidant activity, and provide increased amounts and variants of growth factors And therefore represents a significant advance in the art. As discussed in more detail below, these products and methods can be used in a number of wound healing uses, soft tissue injuries, or osteogenesis restorations.

In one aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane, followed by cryopreservation of the amniotic membrane and subsequent thawing, comprising:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than about 40% of the tissue cells are viable;

B) one or more therapeutic factors that are natural to the amniotic membrane;

C) extracellular matrix that is natural to amniotic membrane; And

D) One or more types of depleted positive immunogenic cells.

In another aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane, followed by cryopreservation of the amniotic membrane and subsequent thawing comprising:

A) a substrate layer comprising a living cell, one or more therapeutic factors, and an extracellular matrix,

B) a surface layer comprising viable cells and one or more therapeutic factors; And

C) one or more types of depleted amounts of functional immunogenic cells,

Over 40% of the combined cells in the matrix and epithelial layers are viable cells.

In one aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing, the at least one tissue component comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane; And

C) Natural extracellular matrix for amniotic membrane;

Amniotic membrane has a depleted amount of functional immunogenic cell types; One or more tissue components are present in an amount effective to provide a therapeutic benefit.

In one aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing, the at least one tissue component comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane; And

C) Natural extracellular matrix for amniotic membrane;

The amniotic membrane has a depleted amount of functional immunogenic cells, and one or more tissue components are present in an amount effective to:

(i) Decreasing the amount and / or activity of proinflammatory cytokines;

(ii) Increasing the amount and / or activity of anti-inflammatory cytokines;

(iii) reducing the amount and / or activity of reactive oxygen species;

(iv) Increasing the amount and / or activity of an antioxidant;

(v) Decreasing the amount and / or activity of the protease;

(vi) Increasing cell proliferation;

(vii) Increasing angiogenesis; And / or

(viii) To increase cell migration.

In another aspect, the disclosure provides a membrane comprising a cryopreserved placenta, followed by cryopreservation of the placenta and subsequent thawing, comprising:

A) Tissue cells, where the placental cells are natural to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the placenta membrane;

C) A natural extracellular matrix for the placenta; And

D) One or more types of depleted amounts of functional immunogenic cells.

In some embodiments of this aspect, the placental membrane comprises amniotic membrane and chorionic membrane.

In some embodiments of this aspect, a depleted amount of functional immunogenic cells produce an immunogenic agent in an amount less than a level sufficient to produce an immune response. In some embodiments, depleted amounts of functional immunogenic cells produce immunogenic agents in amounts less than the detectable limit.

In an embodiment of this aspect, the membrane comprises tissue cells, wherein from about 50% to about 100% of the tissue cells are viable. In some embodiments, about 60% to about 100% of the tissue cells are viable. In a further embodiment, about 70% to about 100% of the tissue cells are viable.

In various embodiments of the foregoing aspects, the membrane may comprise one or more tissue components (e. G., Viable cells, one or more therapeutic agents, and / or adjuvants) in an amount effective to promote any activity in vitro or in vivo (i) / ≪ / RTI > or extracellular matrix).

Various embodiments of the aspects described above may further include a delivery substrate such that the membrane is secured to the delivery substrate.

In embodiments of this aspect, the membrane may be stored for a prolonged period of time prior to subsequent thawing. In some embodiments, the long term time is from about 6 months to about 25 months or more. In these embodiments, the viability of tissue cells is substantially maintained at thawing. In some embodiments, the viability of the tissue cell is substantially maintained for at least about 25 months or more when cryopreserved.

In an embodiment of this aspect, the membrane is thawed and ready for use within the first 30 minutes of the thawing method.

In some embodiments of this aspect, the membranes can be stored in saline until 1 hour after thawing and still retain about 70% viable cells.

In another aspect, the disclosure provides a method of treating a wound in a subject comprising administering a film comprising a cryopreserved amniotic membrane, wherein, after cryopreservation and subsequent thawing, the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

Upon administration, the membrane provides viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to promote one or more of the following:

(i) a decrease in the amount and / or activity of proinflammatory cytokines;

(ii) An increase in the amount and / or activity of anti-inflammatory cytokines;

(iii) reduction in the amount and / or activity of reactive oxygen species;

(iv) An increase in the amount and / or activity of antioxidants;

(v) Reduction in the amount and / or activity of the protease;

(vi) Increased cell proliferation;

(vii) An increase in neovascularization; And / or

(viii) Increased cell migration.

In another aspect, the disclosure provides a method of accelerating wound healing comprising administering a membrane according to any of the aspects and embodiments described herein. In some embodiments, the administering step is effective to promote wound closure for up to 12 weeks after the initial administration phase. In some embodiments, the administering step is effective to promote wound closure 5 to 6 weeks after the initial administration step. In some embodiments, the administering step is effective to promote a reduction in wound size by 50% or more by 28 days after the initial administration step. In an embodiment, the administering step is effective to improve wound closure rate by at least about 44% as compared to standard wound treatment.

In another embodiment, the disclosure provides a method of treating a subject for a wound that is refractory prior to a wound healing treatment, the method comprising administering a membrane to a wound site in accordance with any of the aspects and embodiments herein do. In some embodiments, the administering step is effective to promote wound closure for up to 12 weeks after the initial administration phase. In some embodiments, the administering step is effective to promote wound closure 5 to 6 weeks after the initial administration step. In some embodiments, the administering step is effective to promote a reduction in wound size by 50% or more by 28 days after the initial administration step.

In another aspect, the present disclosure provides a method of treating a chronic wound comprising administering a film comprising a cryopreserved amniotic membrane, wherein the amniotic membrane after cryopreservation and subsequent thawing comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administering step provides a viable therapeutic cell, an extracellular matrix and one or more therapeutic factors in an amount effective to promote the healing of chronic wounds.

In another aspect, the disclosure provides a method of treating an acute wound comprising administering a film comprising a cryopreserved amniotic membrane, wherein the amniotic membrane after cryopreservation and subsequent thawing comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) One or more types of depleted amounts of functional immunogenic cells; The administering step provides a viable therapeutic cell, extracellular matrix and one or more therapeutic factors in an amount that promotes acute wound healing.

In an embodiment of the above aspects relating to wound healing, accelerated wound healing, and / or chronic wound healing, the wound may include a wound, a scar, a burn, a wound, a wound caused by a projectile, a skin wound, a skin wound, Selected from the group consisting of an acute wound, an external wound, an internal wound, a congenital wound, an ulcer, a pressure ulcer, a diabetic ulcer, a water wound, a wound caused by or in addition to a surgical procedure, a venous dermal ulcer, and an avascular necrosis .

In an embodiment of this aspect of the method, the membrane is: (i) a decrease in the amount and / or activity of pro-inflammatory cytokines; (ii) an increase in the amount and / or activity of anti-inflammatory cytokines; (iii) reduction in the amount and / or activity of reactive oxygen species; (iv) an increase in the amount and / or activity of an antioxidant; (v) reducing the amount and / or activity of the protease; (vi) increased cell proliferation; (vii) increased angiogenesis; And / or (viii) an increase in cell migration, either directly or indirectly.

In another aspect, the disclosure provides a method of treating inflammatory eye disease in a subject comprising administering to the subject a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing, the amniotic membrane is:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides viable therapeutic cells, extracellular matrix and one or more therapeutic factors in an amount effective to treat inflammatory eye diseases.

In some embodiments of this aspect, the inflammatory eye disease is a wound or disease characterized by inflammation.

In another aspect, the disclosure provides a method of promoting tissue repair and / or tissue regeneration in a subject, comprising administering to the subject a film comprising amniotic membrane cryopreserved, wherein after cryopreservation and subsequent thawing, silver:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides viable therapeutic cells, extracellular matrix and one or more therapeutic factors in an amount effective to promote tissue repair and / or tissue regeneration.

In some embodiments of this aspect, the method is used in conjunction with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery. In some embodiments, the tissue is a human tissue. In a further embodiment, the human tissue is cartilage, skin, ligament, tendon or bone. In this and various embodiments, the membrane can stimulate tissue regeneration directly or indirectly.

In another aspect, the disclosure provides a method of modulating an inflammatory response comprising administering a film comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing the amniotic membrane is:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

Administration provides viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to reduce the amount and / or activity of the proinflammatory cytokine and / or increase the amount and / or activity of the anti-inflammatory cytokine .

In an aspect, the disclosure provides a method of modulating protease activity, comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing, the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

Administration provides viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to reduce the amount and / or activity of the protease and / or increase the amount and / or activity of the protease inhibitor.

In a further aspect, the disclosure provides an amount of an antioxidant comprising a step of reducing the amount and / or activity of reactive oxygen species (ROS) and administering a membrane comprising amniotic membrane cryopreserved to the wound and / Wherein after cryopreservation and subsequent thawing, the amniotic membrane is: < RTI ID = 0.0 >

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

Administration provides viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to reduce the amount and / or activity of ROS and / or increase the amount and / or activity of the antioxidant.

In another aspect, the disclosure provides a method of increasing neovascularization comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing, the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides an amount of viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to increase angiogenesis.

In another aspect, the disclosure provides a method of increasing cell migration comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing, the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides an amount of living cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to increase cell migration.

In another aspect, the disclosure provides a method of increasing cell proliferation comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing, the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides an amount of living cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to increase cell proliferation.

In an aspect, the disclosure provides a method of preventing or reducing scarring or build up formation in a subject, comprising administering to the subject a film comprising amniotic membrane cryopreserved, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The administration provides viable therapeutic cells, extracellular matrix and one or more therapeutic factors in an amount effective to prevent or reduce scarring or build-up.

In one aspect, the disclosure provides a method of treating or preventing tissue adhesion associated with a surgical procedure comprising administering a film comprising administering a film comprising a cryopreserved amniotic membrane, wherein the cryopreservation and subsequent thawing , Amniotic membrane:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) Depleted amounts of one or more types of functional immunogenic cells;

The membrane is administered to the tissue and provides viable cells, extracellular matrix and / or one or more therapeutic factors in an amount effective to treat or prevent tissue adhesion.

Modulate the inflammatory response, modulate protease activity, decrease the amount and / or activity of reactive oxygen species, increase the amount and / or activity of antioxidants, increase / promote neovascularization, increase cell migration In any of the above aspects of the method of increasing cell proliferation or preventing or reducing scarring or build-up, administration of the membrane may stimulate or induce the method directly or indirectly.

In another aspect, the disclosure provides a kit for the treatment of a wound or tissue defect comprising:

A) A film according to any of the positives and embodiments described herein in a pharmaceutically acceptable container; And

B) Instructions for administering the membrane to treat wounds or tissue defects.

In an embodiment, the kit may further comprise an adduct. In a further embodiment, the adduct is selected from one or more antibiotics, emollients, exfoliants, wetting agents, antioxidants, preservatives, therapeutics, bandages, tools, cutting devices, buffers, thawing media, conditioning media, forceps, .

The foregoing disclosure provides other aspects and embodiments which may become apparent from the following description and from the non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates various refrigeration methods and refrigeration rates for varying amounts of cryopreservation solution. Figs. 1a to 1d are carried out at -80 占 폚 for 30 minutes before refrigerating at 4 占 폚. 1E-1H show direct refrigeration at -80 < 0 &gt; C.
Figure 2 shows the cell processing count (a) and cell viability (b) of the amniotic membrane when stored with varying amounts of cryopreservation solution.
Figure 3 shows the number of cell treatments (A) and cell viability (B) of chorionic villus when stored with varying amounts of cryopreservation solution.
Fig. 4 shows the cryopreservation effect on the cell viability of the villous amniotic membrane.
5 is a view showing the refrigerating time and the refrigeration parameter effect on the cell recovery process (cryopreservation) of amniotic membrane (A) and chorion (B).
Figure 6 shows the effect of refrigeration time and refrigeration parameters on cell viability for amniotic membrane.
7 shows representative images of fresh amniotic membranes (A and C) and chorion (E) as well as cryopreserved amniotic membranes (B and D) and chorion (F) stained with viable / dead strains. The surviving cells are green, while the apoptotic cells are red.
Fig. 8 shows the results of the immunoassay of various placenta membranes (amniotic membrane, chorionic villus (CT), amniotic membrane (ACT), amniotic membrane (AM), chorion (CM) (MLR) assay that measures the expression of IL-2R [alpha] from T-cells stimulated by IL-2R [alpha].
Figure 9 shows IL-2R &lt; alpha &gt; from T-cells stimulated by various placental membranes from the manufacture of intermediate and placental membranes (amniotic membrane (AM), chorionic membrane (CM) Lt; RTI ID = 0.0 &gt; MLR &lt; / RTI &gt; analysis.
10A and 10B show lipopolysacchride (LPS) released from various membrane preparations (amniotic membrane + chorioamnion membrane (ACT), chorionic membrane + trophoblast (CT), trophoblast (T) ) Stimulation TNF- [alpha]. &Lt; / RTI &gt;
Figure 11 shows the expression of IL-2R [alpha] from T-cell stimulation by a high level of TNF- [alpha] secreted chorionic villus (CT).
12 shows images of cultured cells isolated from plastic adherent amniotic membrane (a) and chorion (b). MSCs isolated from human bone marrow aspirate are shown for comparison (c). The possibility of osteogenesis in placenta-derived cells is shown by purple staining for alkaline phosphatase (d).
13 shows the expression of VEGF in new amniotic membrane as compared to frozen-preserved amniotic membrane.
14A is a diagram showing the expression of IFN-2 alpha and TGF- beta 3 in amniotic membrane cell lysate. FIG. 14B shows the expression of bFGF in amniotic membrane (AM) and chorion (CM) extracts.
FIG. 15 is a view showing the expression of BMP-2, BMP-4, PLAB, PlGF (a) and IGF-1 (b) in amniotic membrane cell lysate.
Figure 16 shows the expression of EGF (a), IGFBP1 (b) and adiponectin (c) in amniotic membrane (AM), chorionic membrane (CM) and commercially available products.
Figure 17 shows MMP versus TIMP ratios in amniotic membrane, chorion, and commercially available products.
18 shows the expression of EGF in the amniotic membrane and chorioamnion as measured by ELISA in two distinct placental donors.
Figure 19 shows that cryopreserved amniotic membrane produces a high level of the anti-inflammatory cytokine PGE-2 when exposed to TNF- [alpha].
Figure 20 shows that the cryopreserved amniotic membrane inhibits the release of pre-soluble inflammatory cytokines such as IL-la (a) and TNF-alpha (b) RTI ID = 0.0 &gt; C). &Lt; / RTI &gt;
Figure 21 shows that the amniotic membrane exhibits a statistically significant (* p &lt; 0.05) ability to inhibit MMP activity as evidenced by reduction of the purple dye released.
22 is a view showing the inhibition of elastase by the frozen-preserved amniotic membrane.
23 shows antioxidant ability of frozen-preserved amniotic membrane and ascorbic acid (strong antioxidant);
24 shows that the amphiprotected amniotic membrane can obtain early apoptotic human dermal fibroblast (HDF). Apoptotic cells appear as bright blue dots due to their condensation of chromatin and nuclear fragmentation.
Figure 25 illustrates that the cryopreserved amniotic membrane promotes human Umbilical Vein Endothelial Cells (HUVEC) to form tubes and the number of tubes formed is significantly greater than negative control.
Figure 26 shows a Cell Biolabs 24-well Cytoselect wound healing assay.
Figure 27 shows representative images of human dermal microvascular endothelial cells (HMVEC) treated with 5% conditioned media from a combination of amniotic membrane, chorionic membrane or amniotic membrane / choroidal preparation.
28 is a view showing the promotion of endothelial cell migration by the frozen-preserved amniotic membrane (amniotic membrane);
29 is a view showing the promotion of fibroblast cell migration by the frozen-preserved amniotic membrane (amniotic membrane);
30 is a view showing the promotion of keratinocyte migration due to diseased by frozen-preserved amniotic membrane (amniotic membrane);
Figures 31A-31E illustrate the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 1;
Figures 32A-D illustrate the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 2;
Figure 33 shows that a clinical trial showing complete wound closure results in a placental product (31 of 50 patients, 62.0% wound suture) compared to the control (10 of 47 patients, 21.0% wound suture, p = 0.0001) &Lt; / RTI &gt;
Figure 34 shows Kaplan-Meier analysis (p &lt; 0.0001) showing statistically greater likelihood of complete wound healing during a 12 week evaluation period for placental product compared to the control.
Figure 35 is a plot (p = 0.0001) showing that patients treated with placental products require statistically fewer applications to achieve complete wound closure compared to control arms.
Figure 36 shows Kaplan-Meier analysis showing patients in the control (n = 26) who received placental product treatment up to 12 weeks has a wound closure probability of 67.8%.
37A-37F illustrate a general overview of a method for surgical repair of tendons incorporating the cryopreserved membranes disclosed herein.
38A-38C illustrate a general overview of a method for surgical repair of dry matter incorporating the cryopreserved membranes disclosed herein.

The following definitions apply as used herein:

"Exemplary" (or "for example" or "as an example") means non-limiting examples.

"Chorionic tissue" or "chorionic membrane" means a chorion or a part thereof from a placental tissue, for example, a trocar, mesenchymal stem, or a combination thereof.

"Amniotic membrane tissue" or "amniotic membrane" includes amniotic membrane or part thereof, such as an epithelial layer; Basement membrane; Dense layer; Fibroblast layer; And the amniotic membrane from the intermediate (sponge) layer or a portion thereof.

"Placenta product" or "placenta membrane" means the present placenta products and membranes disclosed and claimed herein. The term includes terms including placenta membranes, cryopreserved placental membranes, cryopreserved chorionic membranes, cryopreserved chorions, and cryopreserved amniotic membranes, and may be used interchangeably with these terms. Placenta products can be used for tissue regeneration and wound restoration.

The term " immunogenicity depleted ", "depleted immunogenic cells" or "depleted immunogenic agents" and placental products or "depleted amounts of functional immunogenic cells & At least one type of " at least one type of immunogenic cell "(e.g., at least one immunogenic cell type) Refers to one or more placental products of the art that are devoid, substantially absent or depleted of CD14 + macrophages, trophic and / or maternal blood cells) and / or immunogenic agents otherwise present in the natural placenta, amniotic membrane, or chorion . A membrane lacking, substantially absent or depleted (similar to the membrane of the techniques described herein) with an immunogenic cell type and / or immunogenicity factor can retain some amount of immunogenic cells / (E.g., in an amount insufficient to produce a functional immune response, in a negligible amount, less than a detectable amount) at an insufficient level to produce a functional response.

An "extracellular matrix" or "ECM" as used herein refers to any one or more components of an extracellular matrix associated with a tissue, such as, for example, placental tissue, including amniotic membrane, chorionic membrane and / Quot; The term refers to soluble / functional therapeutic agents which may be present in the ECM, such as collagen, laminin, fibronectin, hyaluronan, sulfuric acid dermatan sulfate heparin sulfate cordate, decolin and elastin as well as ECM, Proteins, and fragments thereof).

"MSC" means hepatic stem cells and includes fetuses, newborns, adults, or postpartum. "MSC" includes amniotic membrane MSC (AMSC) and chorionic membrane MSC (CMSC). MSCs generally express one or more of CD73, CD70, CD90, CD105 and CD166; Generally, CD45 and CD34 are not expressed. MSCs are differentiated into the mesodermal system (osteogenesis, cartilage origin, and adipocytic).

"Natural cells" or "tissue cells" means cells that are naturally, or are present or endogenous to the placental membrane, i.e. cells that are not exogenously added to the placental membrane, including amniotic membrane or chorion.

"Natural Factor" means a placental membrane factor that is naturally occurring, existing or endogenous to the placental membrane, i.e., a factor that is not exogenously added to the placental membrane.

&Quot; Therapeutic cell "or" beneficial cell "includes cells and components present in the matrix layer and / or epithelial layer of the placenta membrane and includes, for example, MSCs, fibroblasts and / or epithelial cells.

"Therapeutic agent" means a placenta, chorionic membrane, or amniotic membrane-derived factor that promotes wound healing. Therapeutic agents also include molecules that can be classified as cell growth factors / proteins, tissue repair factors / proteins as well as other factors and proteins that generally promote wound healing. Non-limiting examples of therapeutic factors include antimicrobial agents, chemoattractants, remodeling proteins such as proteases and protease inhibitors, immunomodulators, chemokines, cytokines, growth factors and other factors. Therapeutic agents also include factors that promote neovascularization, cell proliferation and epithelialization. Non-limiting examples of such factors include, but are not limited to, TGFα, TGFβ1, TGFβ2, TGFβ3, EGF, HB-EGF, VEGF, VEGF-C, VEGF-D, HGF, PDGF-AA, PDGF- FGF (e.g., FGF-2 / bFGF, KGF, KDG / FGF-7), substrate metalloproteinase (e. G., FGF-2, IGF, IGFBP1, IGFBP2, IGFBP3, adiponectin, (For example, TIMP1 and TIMP2), metalloproteinase inhibitors (for example, TIMP1 and TIMP2), and MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP- , Thrombospondin (e.g., TSP1, TSP2), fibronectin, IL-1RA, NGAL, diphenshine, G-CSF, LIF, IFN2a, PLAB and SDF1b. The term "therapeutic factor" may be used interchangeably with the term "placental factor ".

"Stromal cell" refers to a mixed population of cells (optionally in natural proportions) consisting of neonatal hepatic stem cells and neonatal fibroblasts. Neonatal hepatic stem cells and neonatal fibroblasts are both immunocompromised; Neither expresses a cellular protein that is present in an immunogenic cell type that triggers an immune response.

"Substrate layer" refers to an intra-placental layer that does not contain an epithelial layer.

"In vitro " Describe procedures and / or procedures performed outside of a viable organism including, but not limited to, a culture expansion of a cell (e.g., under tissue culture conditions using an artificial culture medium).

"In vivo" describes experiments and / or procedures performed in an organism, e.g., an animal or human.

"Cryopreserved" or "cryopreserved" or "cryoprotectant" are used interchangeably herein and are materials that help to prevent damage (e.g., cell damage) during the freezing process. Suitable cryopreservatives include dimethylsulfoxide (DMSO), glycerol, glycols, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH) , But are not limited to these. Other cryopreservatives include, for example, polyvinylpyrrolidone, hydroxyethyl starch, polysaccharides, monosaccharides, alginates, trehalose, raffinose, dextran, human serum albumin, phycol, lipoprotein, polyvinylpyrrolidone, For example, one or more non-cell permeable cryopreservatives selected from hydroxyethyl starch, autologous plasma, or combinations thereof. Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4, Sigma-Aldrich (TM) Datasheet).

"Cryopreservation solution" or "cryopreservation medium" refers to a composition comprising at least one cryopreservative. The cryopreservation solution or medium refers to additional components, for example, serum albumin, a pharmaceutically acceptable carrier, buffer, electrolyte solution or saline (e.g., phosphate buffered saline). The cryopreservation solution or medium may be a solution, a slurry, a suspension, or the like.

In general terms, the techniques described herein provide a placental product comprising a manipulated placental tissue. For example, the placenta product may comprise a cryopreserved amniotic membrane, a cryopreserved chorion, and / or a cryopreserved vomiting amniotic membrane. In certain aspects, the cryopreservation method retains a large amount of viable placental cells (i. E., Cells that are natural to the placental tissue (s)) and provides depletion of the immunogenic cells and factors associated with the immunogenic cells. As such, the present disclosure encompasses placental products and cryopreserved amniotic membranes, chorionic membranes and / or chorionic membranes, including combinations of survival cells, therapeutic factors, extracellular matrix and reduced immunogenicity, particularly for use in a number of beneficial therapeutic methods, Or villous amniotic membrane. In certain aspects discussed below, the membranes can be applied to wound or tissue defects and include extracellular matrix capable of direct or indirect induction of changes in the region (e.g., Adaptive medicine). For example, the membrane can be (i) reduced in the amount and / or activity of proinflammatory cytokines; (ii) an increase in the amount and / or activity of anti-inflammatory cytokines; (iii) reduction in the amount and / or activity of reactive oxygen species; (iv) an increase in the amount and / or activity of an antioxidant; (v) reducing the amount and / or activity of the protease; (vi) increased cell proliferation; (vii) increased angiogenesis; And / or (viii) an increase in cell migration, thereby providing a survival cell, therapeutic factor, and extracellular matrix in an amount that can provide or facilitate normal phase of wound healing, such as a chronic wound Lt; RTI ID = 0.0 &gt; healing &lt; / RTI &gt; 3) high levels of proteases and low levels of their inhibitors, as well as high levels of oxidizing agents and low levels of antioxidants that are in balance, as well as a high level of anti-inflammatory cytokines, The features and functionality of the cryopreserved membranes disclosed herein are well suited for such applications because they can include more than one.

In some embodiments, the technique discloses placental products for clinical use, including use in wound healing, such as diabetic foot ulcers, vein leg ulcers, and burns. The method selectively removes essentially all of the potentially immunogenic cells from the placenta membrane while preserving certain cells that play an important role in wound healing.

In some embodiments, the technique selectively discloses a devitalized placenta product. In an embodiment, the placental product may be substantially free of all immunogenic cells. In some embodiments, the membrane does not contain cultured expansion cells.

In some embodiments, the techniques may be combined with at least one therapeutic factor disclosed herein or otherwise known, or a combination of two or more therapeutic factors, such as insulin-like growth factor binding protein 1 (IGFBP1) or adiponectin To provide a placenta product comprising water. In an embodiment, the placental product may comprise epithelial growth factor (EGF) and / or IGFBP1. In an embodiment, the disclosure provides a placental product that can be used to increase anti-inflammatory activity. In an embodiment, the disclosure provides a placental product that can be used to increase antioxidant activity. In embodiments, the disclosure provides placental products that can be used to reduce adhesion and fibrosis, and generally promote scarring activity. In some embodiments, the disclosure provides a placenta product that can be used to increase cell migration. In some embodiments, the disclosure provides a placenta product that can be used to increase the formation of angiostatic structures.

In some embodiments, the technique may be used to selectively immunodeactive cells (e. G., Providing killing or non-immunogenicity) while preserving the viability of certain beneficial cells that are the primary source of factors for promoting wound healing Lt; RTI ID = 0.0 &gt; lyophilized &lt; / RTI &gt;

In some embodiments, the technique discloses a biosynthetic assay to test the immunogenicity of the placenta products produced.

In some embodiments, the technique discloses a placental product that exhibits a treatment cost of MMP versus TIMP similar to that shown in vitro. The present inventors have identified the need for the development of a placental product that exhibits a ratio of MMP-9 to TIMP1 of about 7 to 10 to 1.

In some embodiments, the technique discloses a placental product comprising alpha 2-macroglobulin.

In some embodiments, there is now provided a placenta product capable of inactivating serine protease, cysteine protease, aspartic acid protease, and metalloprotease. In another embodiment, a placenta product is now provided that deactivates serine proteases. In another embodiment, a placenta product is now provided that deactivates the cysteine protease. In a further aspect, a placenta product is now provided that deactivates the aspartic acid protease. In another aspect, a placenta product is now provided that deactivates the metalloprotease.

In some embodiments, the technique discloses a placental product comprising bFGF.

In some embodiments, the technique discloses a placental product that exhibits a desirable MMP to TIMP ratio for wound healing.

In some embodiments, the technique discloses a cell migration assay that can assess the wound healing potential of placental products.

IGFBP1 and adiponectin are among the agents envisioned herein that are important for wound healing. Evaluation of proteins derived from placental products prepared according to the techniques disclosed herein indicates that EGF is one of the major factors secreted in these tissues. In addition, the importance of EGF for wound healing with the high level of EGF detected in amenoriminally disclosed amniotic membranes supports the selection of EGF as an efficacy marker for the evaluation of membrane products prepared for clinical use by this disclosure.

The present technique discloses a cryopreservation procedure for placental products that selectively deplete immunogenic cells and preserve the viability of other beneficial cells (including at least one of the mesenchymal stem cells, epithelial cells and fibroblasts). In some embodiments, the beneficial cells are the primary source of the factor for promoting healing.

Placenta products, their availability, and their immunomodulatory properties are surprisingly enhanced by a deficiency of maternal membranes and selective removal of CD14 + fetal macrophages. Immunocompetence can be demonstrated by any means commonly known by one of ordinary skill in the art and such attestation can be determined by mixed lymphocyte reaction (MLR) and by lipopolysaccharide (LPS) -induced tumor necrosis factor (TNF) -a secretion .

This placenta product optionally contains a basic fibroblast growth factor (bFGF) at a substantial concentration.

The placenta product provides and / or selectively secretes factors that stimulate cell migration and / or wound healing. The presence of such factors can be demonstrated by any conventionally recognized method.

For example, there is a commercially available wound healing assay (Cell Biolabs) and cell migration is performed using human dermal microvascular endothelial cells (HMVEC-d) (Lonza Inc.) Can be evaluated. In one embodiment, the conditioned media from the subject placental product enhances cell migration.

The placental products disclosed herein can be used in a variety of forms including, but not limited to, tendon restoration, cartilage restoration (e.g., femur and tibial plateau), ACL replacement at the tube / bone interface, (E. G., Partial laminar burns, toxic epidermal necrotizing lysis, &lt; RTI ID = 0.0 &gt; (Eg, foot) and vein leg ulcers), surgical wounds, hernia repair, tendon restoration, bladder restoration, osteoarthritis, keloid, tracheal laceration, osteoarthritis, Epithelial defects and repair of the eardrums, or alternatives.

The placental products disclosed herein exhibit one or more of the following properties that are beneficial to the wound healing process:

a. The epithelial cell layer, the approximate number of cells per cm &lt; 2 &gt; of amniotic membrane is about 500 to about 360,000; From about 10,000 to about 360,000; From about 10,000 to about 200,000; Or about 20,000; About 25,000; About 30,000; About 35,000; About 40,000; About 45,000; About 50,000; About 55,000; About 60,000; About 65,000; About 70,000; About 75,000; About 80,000; About 85,000; About 90,000; About 95,000; About 100,000; About 105,000; About 110,000; About 115,000; About 120,000; About 125,000; About 130,000; About 135,000; About 140,000; About 145,000; About 150,000; About 155,000; About 160,000; About 165,000; About 170,000; About 175,000; About 180,000; About 185,000; About 190,000; Or about 195,000.

b. Thick basement membrane (including one or more of I, III, IV collagen, laminin and fibronectin),

c.  A substrate layer;

d. Amniotic membrane having a thickness of about 20 to about 500 탆,

e. Antithrombin activity,

f. Immunogenicity,

g. Cryopreserved / cryopreserved,

h. Amniotic membrane MSC,

i. Analgesic effect

j.  Scar reduction,

k. Reduction of proinflammatory proteins such as IL-l [alpha] and TNF-a,

l. Reduction of anti-inflammatory proteins, such as IL-10,

m. For example, inhibition of CD8 + and CD4 + lymphocyte proliferation and / or immunomodulation by increased amount and / or activity of CD4 + Treg cells,

n. Antibacterial proteins such as dipentin and allantoin (soluble protein),

o. Angiogenesis and re-epithelization, such as angiogenesis and mitogen-promoting factors that promote EGF, HGF and VEGF,

p. Cells that are positive for CD70, CD73, CD90, CD105 and CD166 and negative for CD45 and CD34,

q. Cells expressing HLA-G,

r. Cells that express IDO and FAS ligands that are likely to contribute to immunologic tolerance,

p. Cells with the ability to differentiate into human amniotic epithelial cells (hAEC)

t.  Cells having the ability to differentiate into neurons, hepatocytes and pancreatic cells,

u. The human amniotic membrane with the ability to differentiate into mesodermal system (osteogenesis, cartilage source and adipocyte) and all three embryonic layers - ectoderm (nerve), mesoderm (skeletal muscle, cardiac muscle cell and endothelium) and ectodermal Stem cells (hAMSC),

v. hAMSC express CD49d, which distinguishes them from hAEC,

w.  Stem, or hAMSC positive for the embryonic cytoplasmic marker Oct-4, which plays a role in maintaining pluripotency and self-renewal,

x. HAEC positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, negative for SSEA-4 and non-tumorigenic.

The present inventors have now identified the need for the development of placental products that do not contain culture expanding cells.

The present inventors have now identified the need for the development of a placental product comprising IGFBP1.

The present inventors have now identified the need for the development of a placental product comprising adiponectin.

The present inventors have now identified the need for the development of placental products that exhibit the ratio of protease to protease inhibitor desirable for wound healing.

The present inventors have now identified the need for the development of a method of cryopreserving placental products that preserve the viability of advantageous cells that selectively deplete immunogenic cells from membranes, a primary source of factors for promoting wound healing processes Respectively.

The present inventors have now identified the need for the development of a bioassay to test the immunogenicity of the placenta products produced.

The present inventors have now identified the need for the development of placental products that exhibit an MMP to TIMP ratio similar to that shown in vivo. The present inventors have now identified the need for the development of a placental product that exhibits a ratio of MMP-9 to TIMP1 of about 7 to 10 to 1.

The present inventors have now identified the need for the development of a placental product comprising alpha 2-macroglobulin.

The present inventors have now identified the need for the development of placental products that deactivate serine proteases, cysteine proteases, aspartic acid proteases, and metalloproteases. The present inventors have now identified the need for the development of a captive product that deactivates serine proteases. The present inventors have now identified the need for the development of a captive product that deactivates cysteine protease. The present inventors have now identified the need for the development of a detoxification product that deactivates the aspartic acid protease. The present inventors have now identified the need for the development of a capturing product that deactivates metalloprotease.

The present inventors have now identified the need for the development of placental products containing bFGF.

The present inventors have now identified the need for the development of cell migration assays to assess the potential of placental membrane products.

The present inventors have now identified the need for the development of placental products for wound healing involving hepatic stem cells, epithelial cells and fibroblasts.

Placenta product

summary

In one aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane, followed by cryopreservation of the amniotic membrane and subsequent thawing, comprising:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane;

C) Natural extracellular matrix for amniotic membrane; And

D) One or more types of depleted amounts of functional immunogenic cells.

In another aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane, followed by cryopreservation of the amniotic membrane and subsequent thawing comprising:

A) A substrate layer comprising a surviving cell, one or more therapeutic factors, and an extracellular matrix,

B) A surface layer comprising living cells and one or more therapeutic factors; And

C) One or more types of depleted amounts of functional immunogenic cells.

Over 40% of the combined cells in the matrix and epithelial layers are viable cells.

In another aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing, the at least one tissue component comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane; And

C) Natural extracellular matrix for amniotic membrane;

Amniotic membrane has a depleted amount of functional immunogenic cell types; One or more tissue components are present in an amount effective to provide a therapeutic benefit.

In one aspect, the disclosure provides a film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing, the at least one tissue component comprises:

A) Tissue cells, wherein the tissue cells are native to the amniotic membrane and more than 40% of the tissue cells are viable;

B) One or more therapeutic factors that are natural to the amniotic membrane; And

C) Natural extracellular matrix for amniotic membrane;

The amniotic membrane has a depleted positive functional immunogenic cell type, and one or more tissue components are present in an amount effective to:

(ii) decreasing the amount and / or activity of proinflammatory cytokines;

(ii) increasing the amount and / or activity of anti-inflammatory cytokines;

(iii) reducing the amount and / or activity of reactive oxygen species;

(iv) increasing the amount and / or activity of an antioxidant;

(v) decreasing the amount and / or activity of the protease;

(vi) increasing cell proliferation;

(vii) increasing angiogenesis; And / or

(viii) increasing cell migration.

In another aspect, the disclosure provides a membrane comprising a cryopreserved placenta, followed by cryopreservation of the placenta and subsequent thawing, comprising:

A) tissue cells (the placental cells are natural to the amniotic membrane, and> 40% of the tissue cells are viable);

B) one or more therapeutic factors that are natural to the placental membrane;

C) extracellular matrix that is natural to the placenta; And

D) One or more types of depleted positive immunogenic cells.

In some embodiments of aspects involving placental membranes cryopreserved, the placental membranes include amniotic membrane and chorionic membrane.

In some embodiments of this aspect, a depleted amount of functional immunogenic cells produce an immunogenic agent in an amount less than a level sufficient to produce an immune response. In some embodiments, depleted amounts of functional immunogenic cells produce immunogenic agents in amounts less than the detectable limit. As described herein, a depleted amount of functional immunogenic cells can be selected from any one or more of maternal blood cells, neonatal blood cells, umbilical blood cells, tissue macrophages, and trophoblasts. In a further embodiment, the depleted amount of functional immunogenic cells comprises one or more tissue macrophages. Tissue macrophages may have tissue macrophages that are selected from the group consisting of any characterized macrophage, e. G., CD11b +, CD14 +, CD18 +, CD40 + and CD86 +, and / or combinations thereof. In a further embodiment, a depleted amount of functional immunogenic cells is treated with one or more immunogenic agents (e. G., TNF-a, and / or described herein or otherwise disclosed) in an amount less than a sufficient level to produce an immune response RTI ID = 0.0 &gt; immunogenicity &lt; / RTI &gt; In further embodiments, the immunogenicity factor may be produced in an amount that is negligible or below the detectable limit. In some embodiments, less than 10% of the viable cells are functional immunogenic cells (e. G., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% % Or less than 1%).

In an embodiment of this aspect, the membrane comprises tissue cells, wherein from about 50% to about 100% of the tissue cells are viable. In some embodiments, about 60% to about 100% of the tissue cells are viable. In a further embodiment, about 70% to about 100% of the tissue cells are viable. In a further embodiment, the surviving cells may comprise hepatic stem cells (MSC), fibroblasts and / or epithelial cells.

In various embodiments of the foregoing aspects, the membrane may comprise one or more tissue components (e. G., Viable cells, one or more therapeutic agents, and / or adjuvants) in an amount effective to promote any activity in vitro or in vivo (i) / &Lt; / RTI &gt; or extracellular matrix).

Experiments and / or procedures performed outside of an in vitro viable organism (e.g., under tissue culture conditions using an artificial culture medium) are described. Experiments and / or procedures performed in vivo in organisms such as animals or humans are described.

Various embodiments of the aspects described above may further include a delivery substrate such that the membrane is secured to the delivery substrate.

In embodiments of this aspect, the membrane may be stored for a prolonged period of time prior to subsequent thawing. In some embodiments, the long term time may be from about 6 months to at least about 36 months, alternatively from about 6 months to at least about 24 months, including other monthly and day variations thereof for the various time periods described herein Alternatively from about 6 months to about 25 months, alternatively from about 6 months to at least about 12 months, alternatively from about 6 months to about 10 months, alternatively from about 6 months to about 25 months, Alternatively from about 3 months to about 6 months, alternatively from about 1 month to about 3 months. In these embodiments, the viability of tissue cells is substantially maintained at thawing. In some embodiments, the viability of the tissue cells is substantially maintained for at least about 24 months when cryopreserved.

In an embodiment of this aspect, the membrane may be thawed and may be ready for use within the first 30 minutes of the thawing method as described herein, or as modified from generally known methods.

In some embodiments of this aspect, the membranes can be stored in saline until 1 hour after thawing and still retain about 70% viable cells.

One embodiment of the present technology provides a placenta product comprising a cryopreservation solution and a amniotic membrane, wherein the amniotic membrane comprises a natural therapeutic cell and a natural therapeutic factor, wherein the cryopreservation solution comprises a quantity of a cryopreservative effective to provide a cryopreserved product . According to this embodiment, the amniotic membrane is substantially free of at least one or both of the following immunogenic cell types (CD14 + macrophages and maternal blood cells).

In one embodiment, the amniotic membrane comprises one or more layers that represent the structure of the natural amniotic membrane (e.g., not homogenized or treated with collagenase).

In one embodiment, the placenta membrane is suitable for dermal application to the wound.

By the teachings provided herein, one of ordinary skill in the art can now produce the placenta product or membrane. The present disclosure provides a method of manufacture that produces technical features of the present placenta product. Thus, in one embodiment, the placental product is produced by the steps taught herein. The placenta product is not limited to the products produced by the methods taught herein. For example, the product of the present technology may include selection steps; For example, by methods that rely on the selection steps for the formulation with the described technical features and superior properties.

The placenta product may include one or more of the following technical characteristics:

a. Survivable therapeutic natural cells can differentiate into cells of one or more lines (e.g., bone formation, adipocyte and / or cartilage lineage)

b. Natural therapeutic factors include IGFBP1,

c. Natural therapeutic factors include adiponectin,

d. Natural therapeutic agents include [alpha] 2-macroglobulin,

e. Natural therapeutic agents include bFGF,

f. Natural therapeutic factors include EGF,

g. Natural therapeutic factors include specific amounts of MMP-9 and TIMP1,

h. Natural therapeutic agents include MMP-9 and TIMP1 at a ratio of about 7 to about 10,

i. Placenta products do not contain cultured expanded cells,

j. The cryopreservation solution contained about May be present in an amount greater than 15 mL,

k. Cryopreservatives include DMSO,

l. The cryopreservation solution further comprises albumin, optionally albumin is human serum albumin (HSA)

m. Cryopreservation includes DMSO and albumin (e.g., HSA)

n. About 5,000 to about 500,000 cells / cm2, about 5,000 to about 240,000 cells / cm 2 Or from about 20,000 to about 60,000 cells / cm2 / RTI &gt;

o. Amniotic membrane comprises amniotic membrane at least: about 2,000, or about 2,400, or about 4,000 ^, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells /

p. The amniotic membrane comprises amniotic membrane of about 2,000 to about 15,000 stromal cells / cm 2,

q. About 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4%, or about 90%, or about 92.5%, or at least about 100% The cells are alive after a freeze-thaw cycle,

r. Wherein about 40% to about 92.5% of the stromal cells are alive after the freeze-thaw cycle,

p. The amniotic membrane has a thickness of about 20 탆 to about 500 탆; From about 20 [mu] m to about 200 [mu] m; Or from about 20 [mu] m to about 100 [mu] m.

t. Is secreted below any of the following: a 2 cm x 2 cm piece of tissue product is placed in the tissue culture medium and the tissue product is exposed to the bacterial lipid polysaccharide for about 20 to about 24 hours, Cm2, 225 pg / cm2, 100 pg / cm2, or 70 pg / cm2 The following TNF-alpha,

u. After chilling, cryopreservation, and thawing, they are secreted below any of the following: Place a 2 cm x 2 cm piece of tissue product in a tissue culture medium and expose the tissue product against the bacterial lipid polysaccharide for about 20 to about 24 hours , 350 pg / cm 2, 225 pg / cm 2, 100 pg / cm 2, or 70 pg / cm 2 into the tissue culture medium The following TNF-alpha,

v. Further comprising chorionic villi,

w. The amniotic membrane contains a layer of epithelial cells,

x. Further comprising a chorionic membrane, wherein the amniotic membrane and chorion are associated with a natural stereotactic arrangement,

y. In addition, the amniotic membrane and chorion are not attached to the native stroma,

z. Further comprising a chorion, wherein the chorion comprises about 2 to about 4 times more stromal cells for amniotic membrane,

aa. Does not contain chorion;

bb. Wherein the chorionic villus comprises about 2 to about 4 times more stromal cells for amniotic membrane,

cc. It is suitable for applying dermis to wound.

cell

According to the technique, the placental product comprises amniotic membrane naturally treated cells. The cells include one or more of MSC, fibroblasts, and epithelial cells.

In one embodiment, the naturally treated cell comprises a viable MSC.

In one embodiment, the naturally treated cells comprise viable fibroblasts.

In one embodiment, the naturally-treated cells comprise viable epithelial cells.

In one embodiment, the naturally treated cells comprise viable MSCs and viable fibroblasts.

In one embodiment, the naturally-treated cells comprise viable MSCs, viable fibroblasts and viable epithelial cells.

In one embodiment, a viable therapeutic natural cell is a viable cell capable of differentiating into cells of one or more lines (e.g., bone formation, adipocyte and / or cartilage lineage).

In one embodiment, the placental product comprises about 500 to about 360,000 cells / Or from about 40,000 to about 90,000 cells / cm2.

In one embodiment, the placenta product is at least about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000, or about 180,000 substrate cells / Includes placenta products.

In one embodiment, the placental product comprises a placental product of from about 2,000 to about 15,000 stromal cells / cm 2.

In one embodiment, the placental product comprises at least about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4%, or about 90% %, Or about 100% of the stromal cells are viable after the freeze-thaw cycle.

In one embodiment, the placental product comprises stromal cells, wherein from about 40% to about 100% of the stromal cells are alive after the freeze-thaw cycle, alternatively from about 40% to about 99.9% of the stromal cells are alive , Alternatively from about 70% to about 99% of the stromal cells are viable.

In one embodiment, the placental product comprises less than about 1% CD14 + macrophages / total cells.

In one embodiment, the amniotic membrane of the placental product comprises from about 2 to about 4 times less stromal cells as compared to the chorion from the same placenta.

In one embodiment, the placental product further comprises a chorionic membrane containing from about 2 to about 4 times more stromal cells per amniotic membrane.

In one embodiment, the placental product comprises MSC in an amount up to about 70% of the total number of cells in the amniotic membrane product of the amniotic membrane. In some embodiments, the product is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, from about 1% to about 10% Or MSC in an amount of about 3% to about 100%. In some embodiments of the total number of MSCs in the amniotic membrane product, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the MSC is viable after the freeze-thaw cycle.

In one embodiment, the placental product comprises fibroblasts in an amount up to about 100% of the total number of cells in the amniotic membrane product. In some embodiments, the product is present in an amount of about 1%, about 20%, about 5% to about 15%, at least about 1%, at least about 2%, at least about 3% Including fibroblasts in an amount of 4%. In some embodiments of the total number of fibroblasts in the amniotic membrane product, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the fibroblasts are viable after the freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product is about 5% to about 100%, about 5% to about 50%, about 5% to about 30%, about 10% to about 30% %, About 15% to about 25%, at least about 5%, at least about 10%, or at least about 15%. Alternatively, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the stromal cells are viable after the freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product is about 60% to about 100%, about 60% to about 90%, about 70% to about 90%, about 40% to about 90% %, About 50% to about 90%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the epithelial cells. Alternatively, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the epithelial cells are viable after the freeze-thaw cycle.

In embodiments, the total number of functional macrophages is less than about 10% (e.g., about 10%, about 9%, about 8%, about 7%, about 6%, about 5% , Less than about 2%, or less than about 1% of viable cells.

In one embodiment, the amniotic membrane of the placental product comprises fibroblast and MSC at a ratio of about 4: 1 to about 1: 1, or about 3: 1 to about 3: 2, or about 2:

In one embodiment, the amniotic membrane of the placental product is an MSC in an amount of at least about 1,000 cells / cm2 ^, at least about 2,000 cells / cm2, about 1,000 to about 5,000 cells / cm2, or about 2,000 to about 5,000 cells / . Alternatively, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the MSCs are viable after the freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product is a fibroblast cell in an amount of at least about 2,000 cells / cm2, at least about 4,000 cells / cm2, about 2,000 to about 9,000 cells / cm2, or about 2,000 to about 9,000 cells / . Alternatively, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the fibroblasts are viable after the freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product has at least about 4,000, at least about 8,000 cells / cm2, at least about 4,000 to about 18,000 cells / Or stromal cells in an amount of from about 4,000 to about 18,000 cells / cm2. Alternatively, at least about 40%, about 50%, about 60%, about 70%, or about 100% of the stromal cells are viable after the freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises at least about 10,000 cells / cm2, at least about 20,000 cells / cm2, at least about 32,000 cells / cm2, between about 10,000 and about 72,000 cells / At least about 40%, about 50%, about 60%, about 70%, or about 100% of the epithelial cells comprise the epithelial cells It is viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placenta product is about 3,000 cells / cm2 , Less than about 1,000 cells / cm2, or less than about 500 cells / cm2.

In one embodiment, the placental product comprises a layer of epithelial cells.

In one embodiment, the placental product comprises a chorionic membrane, but is substantially free of hypertrophy.

In one embodiment, the placental product is substantially free of functional CD14 + macrophages.

In one embodiment, the placental product is substantially free of maternal blood cells.

In one embodiment, the placental product is substantially free of nutrient, functional CD14 + macrophages, and maternal blood cells. Alternatively, the placental product comprises viable stromal cells. Optionally, the placenta product comprises a viable MSC. Alternatively, the placental product comprises viable fibroblasts. Alternatively, the placental product comprises viable epithelial cells. Alternatively, the placental product comprises viable MSCs, fibroblasts and epithelial cells.

In one embodiment, the placental product comprises chorion, but is substantially free of maternal blood cells.

In one embodiment, the placental product comprises chorion but is substantially free of maternal blood cells and / or trophoblast cells.

In one embodiment, the placental product is substantially free of cultured expanded cells.

Placenta factor

According to the present technique, placental products include amniotic membrane natural therapies as defined and otherwise described above in various aspects and embodiments, including embodiments. Table 10 provides a list of the therapeutic factors tested and their function.

In one embodiment, the factor comprises one or more of the following: IGFBP1, adiponectin, a2-macroglobulin, bFGF, EGF, MMP-9 and TIMP1. Alternatively, the factor is present in a quantity / cm2 substantially similar to the natural amniotic membrane or layer thereof (e.g., +/- 10% or 20%).

In one embodiment, the factor includes VEGF, PDFG, HGF, SDF-1, KGF, IGFBP1, adiponectin, a2-macroglobulin, bFGF, EGF, MMP-9 and TIMP1. Optionally, the factor is present in a ratio substantially similar to its natural amniotic membrane. Alternatively, the factor is present in a quantity / cm2 substantially similar to the natural amniotic membrane or layer thereof (e.g., +/- 10% or 20%).

In one embodiment, the factor comprises MMP-9 and TIMP1 at a ratio of about 7 to about 10 (e.g., about 7). Alternatively, the factor is present in a quantity / cm2 substantially similar to the natural amniotic membrane or layer thereof (e.g., +/- 10% or 20%).

In one embodiment, the factor includes one or more of the factors listed in Table 10 (e.g., the majority or all). Alternatively, the agent is present in a ratio substantially similar to the natural amniotic membrane or layer thereof. Alternatively, the factor is present in a quantity / cm2 substantially similar to the natural amniotic membrane or layer thereof (e.g., +/- 10% or 20%).

In one embodiment, the placental product comprises substantially less than TNF-a / cm &lt; 2 &gt; than the natural amniotic membrane or layer thereof, respectively.

In one embodiment, the placental product secretes substantially less than TNF-a / cm &lt; 2 &gt; than the natural placenta product or layer thereof, respectively.

In one embodiment, the placental product is secreted below any of the following: placing a 2 cm x 2 cm piece of tissue product in a tissue culture medium and exposing the tissue product against bacterial lipid polysaccharide for about 20 to about 24 hours ML, 350 pg / mL or 280 pg / mL TNF-a into tissue culture medium.

In one embodiment, after refrigeration, cryopreservation, and thawing, the placenta product is secreted below any of the following: a 2 cm x 2 cm piece of tissue product is placed in the tissue culture medium and maintained for about 20 to about 24 hours When exposed to tissue products for lipid polysaccharides, 420 pg / mL, 350 pg / mL, or 280 pg / mL TNF-α into tissue culture medium.

In one embodiment, the placental product further comprises an exogenously added inhibitor of TNF- [alpha]. Optionally, the inhibitor of TNF- [alpha] is PGE2.

In one embodiment, the product can be treated with an antibiotic or antibiotic cocktail. One non-limiting example of an antibiotic cocktail includes gentamicin sulfate, vancomycin HCl, and amphotericin B.

Configuration

The placenta product of the present technology comprises one or more layers that represent the structure of the natural amniotic membrane. By the teachings provided herein, one of ordinary skill in the art will recognize a placental layer that exhibits a native structure, for example, a layer that is not homogenized or that has not been treated with collagenase or other enzymes that substantially destroy the layer.

In one embodiment, the placental product comprises a substrate layer having the native structure of the amniotic membrane.

In one embodiment, the placental product comprises a basement membrane having the natural structure of the amniotic membrane.

In another embodiment, the placental product comprises an epithelial layer having the native structure of the amniotic membrane.

In one embodiment, the placental product comprises a matrix layer and a layer of epithelial cells having the native structure of the amniotic membrane.

In one embodiment, the placenta product or amniotic membrane thereof has a thickness of from about 20 탆 to about 500 탆; From about 20 [mu] m to about 200 [mu] m; Or from about 20 [mu] m to about 100 [mu] m.

In one embodiment, the placental product comprises a chorionic membrane, but is substantially free of hypertrophy. In one embodiment, the placental product comprises a basement membrane having a native structure of the chorion, and the chorion membrane is substantially free of hypertrophy. Optionally, the parent moiety upon contact with the chorionic membrane comprises a fragment of an extracellular matrix protein. Alternatively, the placental product has been treated with a dispase (e. G., Dispase II) and a substantial portion of the extracellular matrix protein fraction comprises terminal leucine or phenylalanine.

In one embodiment, the placental product further comprises a chorionic membrane. Alternatively, the amniotic membrane and chorion within the placenta product are associated with native conformations. Alternatively, the amniotic membrane and the chorionic membrane are not attached to each other in a native steric configuration.

In one embodiment, the placenta product does not contain a chorion.

Formulation

According to the present technique, the placenta product can be formulated with a cryopreservative solution.

In one embodiment, the cryopreservation solution comprises one or more cell-permeable cryopreservants, one or more non-cell-permeable cryopreservants, or a combination thereof.

Optionally, the cryopreservation solution comprises one or more cell-permeable cryopreservation agents selected from DMSO, glycerol, glycols, propylene glycol, ethylene glycol, or combinations thereof.

Optionally, the cryopreservation solution comprises one or more cell-permeable cryopreservatives selected from polyvinylpyrrolidone, hydroxyethyl starch, polysaccharides, monosaccharides, sugar alcohols, alginates, trehalose, raffinose, dextran, .

Another example of a useful cryoprotectant is described in "Cryopreservation" (BioFiles, Volume 5, Number 4 Sigma-Aldrich (TM) Data Sheet).

In one embodiment, the cryopreservation solution comprises a cell-permeable cryopreservation agent, with the majority of cell-permeable cryopreservation agents being DMSO. Optionally, the cryopreservation solution does not contain a substantial amount of glycerol.

In one embodiment, the cryopreservation solution comprises DMSO. Optionally, the cryopreservation solution does not contain a large amount of glycerol. Optionally, the cryopreservation solution does not contain a substantial amount of glycerol.

In one embodiment, the cryopreservation solution comprises additional components such as albumin (e.g., HSA or BSA), electrolyte solution (e.g., Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation solution comprises between 1 vol% and about 15 vol% albumin and between about 5 vol% and about 20 vol% cryopreservation (e.g., about 10 vol%). Optionally, the cryopreservative comprises DMSO. In some embodiments, the cryopreservation solution comprises from about 5% to about 100% cryopreservation agent, alternatively from about 5% to about 20%.

In one embodiment, the placenta product is formulated into a cryopreserved solution of about 20 mL or greater than about 50 mL. Optionally, the cryopreservation solution comprises at least one cryopreservative (or cryoprotectant). In some aspects, at least one cryoprotectant comprises DMSO (e.g., if one or more cryopreservants are present, DMSO is found in a large amount of total cryopreservation agent). Optionally, the cryopreservation solution does not contain a substantial amount of glycerol.

In some embodiments, the composition comprises a cryopreservation solution. The cryopreservation solution may optionally be added to a container containing the placenta product as a film-securing composition (for example, on nitrocellulose). Preferably, a sufficient amount of cryopreservation solution is added to protect the membrane during the subsequent freezing step. Injection of the membrane by the cryopreserved solution maintains the viability of the cells contained in the membrane. Although suitable cryopreservation solutions are known in the art, in one embodiment, the cryoprotectant is stored in a cryopreserved solution comprising one or more cell-permeable cryopreservation agents, one or more non-cell permeable cryopreservation agents, or a combination thereof .

Suitable cryopreservatives include, but are not limited to, DMSO, glycerol, glycols, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH) . In some embodiments, the cryopreservation solution is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, polysaccharide, monosaccharide, alginate, trehalose, raffinose, dextran, human serum albumin, phycol, lipoprotein, polyvinylpyrrolidone, One or more non-cell permeable cryopreservatives selected from hydroxyethyl starch, autologous plasma, or a combination thereof. Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4, Sigma-Aldrich (TM) Datasheet).

For example, a suitable cryopreservation solution may be stored in an amount of at least about 0.001 vol% to 100 vol%, suitably about 2 vol% to about 20 vol%, preferably about 5 vol% to about 10 vol% For example, DMSO. In some examples, the cryopreservation solution comprises at least about 2% cryopreservation agent (e. G., DMSO). Additionally, the cryopreservation solution may comprise serum albumin or other suitable protein. In some embodiments, the cryopreservation solution comprises from about 1% to about 20% serum albumin or other suitable protein, alternatively from about 1% to about 10%. Serum albumin or other suitable protein is provided to stabilize the membrane during the freeze-thaw process and to maintain viability by reducing damage to the cells. The serum albumin may be human serum albumin or bovine serum albumin. The cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffered saline.

During the cryopreservation process, the container may be filled with a sufficient amount of cryopreservation solution to cover the placental membrane. The amount of cryopreservation solution may inevitably depend on a number of factors including, for example, the type of container used and the size of the membrane to be preserved as well as the seal. The lower the amount of cryoprotectant solution that is necessary to (or over) the composition / device, the faster the composition can be thawed. Thus, it may be desirable to use the minimum amount of cryopreservation solution allowed for the upper range of the membrane without compromising cell viability during cryopreservation. In addition, the smaller the membrane used and the smaller the vessel, the less cryopreservation solution may be used.

In some embodiments, a solution containing a cryopreserve solution in an amount from about 7 mL to about 50 mL, alternatively from about 10 mL to about 50 mL, alternatively from about 15 mL to about 50 mL, alternatively from about 15 mL to about 25 mL A bag can be used. In one preferred embodiment, about 15 mL of the cryopreserved solution is added to the container or bag. The amount of cryopreservation solution may be sufficient to completely immerse the membrane. The amount will depend on the size of the bag used and the size of the cryopreserved film. If a small bag is used with a small membrane (e.g., smaller than a 2 cm x 2 cm membrane), a cryopreservation solution of from about 3 mL to about 10 mL, alternatively from 3 mL to about 7 mL, may be used.

In some embodiments, a container containing from about 7 mL to about 50 mL, alternatively from about 5 mL to about 20 mL, alternatively from about 7 mL to about 20 mL, alternatively from about 7 mL to about 15 mL is used. The amount of cryopreservation solution may be sufficient to sufficiently immerse the membrane in the vessel. The amount will depend on the size of the container used and the size of the cryopreserved membrane.

In some embodiments, the amount of cryopreservation solution is sufficient to protect the cells during cryopreservation and subsequent thawing procedures. In some embodiments, at least 70% cell viability is maintained after freeze-thaw. In some aspects, at least 75% cell viability is maintained, alternatively about 80% cell viability is maintained, alternatively 85% cell viability is maintained, and alternatively about 90% cell viability is maintained , Alternatively about 95% cell viability is maintained, and alternatively about 100% cell viability is maintained. In some embodiments, the viability of the membrane is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 75%, at least 78%, at least 80% 85%, at least 88%, at least 89%, at least 90%, at least 92%, and a percentage therebetween.

In some embodiments, the amount of cryopreservation solution is sufficient to protect the structural, constitutive and / or 3-D structure of the membrane. In these examples, the cryopreservation solution contains cell-permeable cryopreservation agent in an amount of from 0.01% to about 100%, alternatively from about 2% to about 100%. In some embodiments, the cryopreservation solution contains polysaccharides or monosaccharides.

In one embodiment, the placenta product is placed on nitrocellulose paper.

In one embodiment, the placenta, amniotic membrane and / or chorionic membrane may be cut into a plurality of sections. Optionally, the membrane will be of any suitable size depending on the membrane type and the particular end use or usage of the membrane, and can be customized. Suitable sizes of the membrane are about 1.5 cm x about 1.5 cm, about 2 cm x about 2 cm, about 3 cm x about 3 cm, about 4 cm x about 4 cm, about 5 cm x about 5 cm, about 6 cm x about 6 cm About 7 cm x about 7 cm, about 8 cm x about 8 cm, about 7.5 cm x about 15 cm, about 1.5 cm x about 2 cm, about 1.5 cm x about 3 cm, about 2 cm x about About 3 cm x about 4 cm, about 2 cm x about 5 cm, about 3 cm x about 5 cm, about 4 cm x about 5 cm, about 5 cm x about 7 cm, about 5 cm x about 10 cm , About 5 cm x about 15 cm, about 7.5 cm x about 15 cm, but is not limited thereto and includes any variation or size and range in increments of 0.1 cm to 1 cm.

Cryopreserved membranes have surprisingly long shelf life or stability and retain viable cells when frozen for long periods of time. The cryopreserved product is preferably stored at a temperature of about -20 ° C to about -196 ° C for more than two years (eg, at least 40%, 50%, 60%, or 70% For example, from about -45 to about -80 &lt; 0 &gt; C). In some aspects, the cryopreserved membrane may be a viable cell (e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80% viable cells, At least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 24 months, at least about 12 months, at least about 12 months, at least about 12 months, At least about 25 months, at least about 36 months, at a temperature of about-20 C to about -196 C (e.g., about -45 C to about -80 C).

In another aspect, the disclosure provides a kit for the treatment of a wound or tissue defect comprising:

A) A film according to any of the positives and embodiments described herein in a pharmaceutically acceptable container; And

B) Instructions for administering the membrane to treat wounds or tissue defects.

In an embodiment, the kit is selected from one or more antibiotics, an emollient, a keratolytic agent, a humectant, an antioxidant, a preservative, a therapeutic, a bandage, a tool, a cutting device, a buffer, a thawing medium, a conditioning medium, a forceps, Adducts.

Produce

summary

Placenta products of the present technology can be prepared from the placenta in any suitable manner that provides the technical features taught herein. According to the technique, the placenta product comprises amniotic membrane which is at least immunocompetent.

In one embodiment, the placenta product is produced by a method comprising the steps of:

a. The placenta obtaining step,

b. Selectively depleting the placental membrane immunogenicity; And

c. The placental membrane is cryopreserved.

Alternatively, the method includes removing maternal blood cells from the placenta membrane, for example, by hemolyzing red blood cells, removing blood clots, or a combination thereof.

Optionally, the method comprises treating the placental membrane with one or more antibiotics.

Alternatively, the method comprises selectively depleting CD14 + macrophages, as evidenced by a substantial reduction in LPS stimulation of TNFa release.

Optionally, lyophilizing the placenta membrane comprises freezing the membrane in the cryopreservation solution comprising one or more cell-permeable cryopreservation agents, one or more non-cell-permeable cryopreservation agents, or a combination thereof.

Optionally, lyophilizing the placenta membrane comprises Optionally, depleting CD14 + macrophages as demonstrated by substantial reduction in LPS stimulation of TNF [alpha] release by placing at a temperature of about 2 [deg.] C to about 8 [deg.] C for a period of time, followed by freezing at -80 [ .

Optionally, the method comprises maintaining an epithelial layer of amniotic membrane.

Optionally, the method comprises removing the chorion or part thereof. Optionally, the method comprises removing the nutrient film from the chorionic membrane while maintaining a substrate layer of the chorion.

Exemplary placental membranes of the present technology may be made or provided with bandages or wound dressings.

Immunocompetence and selective depletion

In one embodiment, the placental product or membrane of the present technique is immunocompetent. Immunocompetence may be performed by any optional depletion step that removes immunogenic cells or factors or immunogenicity (or amniotic membrane thereof) from the placenta.

In one embodiment, the placental product becomes immunocompetent by selectively depleting functional immunogenic cells. The placenta may become immunocompetent by selectively removing immunogenic cells from the placenta product (or amniotic membrane thereof) while maintaining therapeutic cells. For example, immunogenic cells can be removed by killing immunogenic cells or by placental purification from them.

In one embodiment, the placental product becomes immunocompetent by selectively depleting the trophic membrane, e.g., by removal of the trophic layer.

In one embodiment, the placental product becomes immunocompetent by substantial reduction of CD14 + macrophage, optionally LPS stimulation of TNFa release, or by selective depletion of functional CD14 + macrophages, as evidenced by MLR analysis.

In one embodiment, the placental product becomes immunocompetent by selective depletion of maternal blood cells.

In one embodiment, the placental product is rendered immunocompetent by selective depletion of functional CD14 + macrophages, trophic and maternal blood cells.

In one embodiment, the placental product is immunoconjugated by a substantial reduction of LPS stimulation of the trophoblast and / or CD14 + macrophage, optionally TNFa release, or by selective depletion of functional CD14 + macrophages, as evidenced by MLR analysis .

Nutrient  remove

In one embodiment, immunoconjugation (or selective depletion) is performed by elimination or depletion of the nutrient from the placenta product. Removal of the nutrient film from the chorion is performed while the substrate layer of the chorion is maintained. Surprisingly, such placental products have one or more of the following outstanding characteristics:

a. Substantially non-immunogenic;

b. Provides remarkable healing; And

c. Provides improved therapeutic efficacy.

In one embodiment, the nutrient film is removed while the substrate layer of the chorion is maintained.

The nutrient film can be removed in any suitable manner that substantially reduces the nutrient content of the placenta product. In one embodiment, the nutrient film is selectively removed. Alternatively, the nutrient film is selectively removed or otherwise removed without removing a substantial portion (e.g., MSCs, therapeutic factors, extracellular matrix, etc.) of one or more therapeutic ingredients from the chorion. Alternatively, a majority (e.g., substantially all) of the nutrient film is removed.

One way to remove the trophoblasts is to treat the placenta (e.g., chorionic membrane or amniotic membrane-chorion) using digestive enzymes such as dispase (e.g., Dyspase II) and isolate the nutrient from the placenta . Optionally, the step of separating includes mechanical separation such as peeling or scraping. Optionally, the scraping includes scraping using a soft mechanism such as a finger.

A method of removing the trophoblast comprises treating the chorion with dispase for about 30 to about 45 minutes to separate the herbicidal film from the placenta. Optionally, the dispase is provided in a solution of less than about 1% (e.g., about 0.5%). Optionally, the step of separating includes mechanical separation such as peeling or scraping. Optionally, the scraping includes scraping using a soft mechanism such as a finger.

A useful method for removing the supernatant from the placenta (e.g., chorion) is described by Portmann-Lanz et al. (&Quot; Isolation and characterization of mesenchymal cells from human fetal membranes "; Journal of Obstetrics and Gynecology (2006) 194, 664-73), Journal of Obstetrics and Gynecology Tissue Engineering And Regenerative Medicine 2007; 1: 296-305. &Quot; and Concise Review: Isolation and Characterization of Cells from Human Primary Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells.

In one embodiment, the nutrient film is removed prior to cryopreservation.

Removal of macrophages

In one embodiment, the functional macrophages are depleted of placental products or eliminated. Surprisingly, such placental products have one or more of the following outstanding characteristics:

a. Substantially non-immunogenic;

b. Provides remarkable healing; And

c. Provides improved therapeutic efficacy.

The functional macrophages can be eliminated in any suitable manner that substantially reduces the macrophage content of the placental product. Alternatively, the macrophage is selectively removed or otherwise removed without removing a substantial portion (e.g., MSCs, therapeutic factors, etc.) of one or more therapeutic ingredients from the placenta. Alternatively, a majority (e. G., Substantially all) macrophages are removed.

One method of removing immune cells, such as macrophages, involves immune cell killing by rapid freezing rates, such as 60-100 ° C / min. Another method of removing immune cells is by keeping the cells at about 2 ° C to about 8 ° C for a period of time and then by freezing the immune cells at a rate of about 1 ° C per minute (eg, at about -20 ° C ) &Lt; / RTI &gt;

Although immune cells can be removed by fast freezing rates, this method may also be detrimental to therapeutic cells such as stromal cells (e.g., MSC). The inventors have found that the placental membrane is maintained at said refrigeration temperature (e.g., at 2 to 8 DEG C) for a period of time at about 2 DEG C to about 8 DEG C (e.g., for at least about 10 minutes, Incubation) followed by selective quenching of CD14 + macrophages by freezing the placenta (e.g., incubation at -80 ° C ± 5 ° C). Optionally, the freezing step includes freezing at a rate of less than 10 ° / minute (eg, less than about 5 ° / minute, such as less than about 1 ° / minute).

In one embodiment, the step of placing the placental membrane at about 2 DEG C to about 8 DEG C includes cryopreserving solution (e.g., DMSO ) Containing the &lt; RTI ID = 0.0 &gt; placenta &lt; / RTI &gt; Optionally, the freezing step comprises reducing the temperature at a rate of about 1 DEG / min. Optionally, the freezing step includes freezing at a rate of less than 10 ° / minute (eg, less than about 5 ° / minute, eg, about 1 ° / minute).

In one embodiment, the step of disposing the placental membrane at about 2 DEG C to about 8 DEG C comprises cryopreserving solution (e.g., at about-10 to 15 DEG C) (for example, at 2-8 DEG C) Containing DMSO): 10 min, 20 min, 30 min, 40 min or 50 min. In another embodiment, the step of disposing the placental membrane at about 2 DEG C to about 8 DEG C includes placing the placental membrane in a cryopreserved solution (e.g., at a temperature of about -10 to 15 DEG C For example, DMSO): 10 to 120, 20 to 90 minutes, or 30 to 60 minutes. The freezing step includes freezing at a rate of less than 10 / minute (e.g., less than about 5 / minute, such as less than about 1 / minute).

Removal of maternal blood cells

In one embodiment, maternal blood cells are depleted of placental products or removed from placental products. Surprisingly, such placental products have one or more of the following outstanding characteristics:

a. Substantially non-immunogenic;

b. Provides remarkable healing; And

c. Provides improved therapeutic efficacy.

The maternal blood cells may be removed in any suitable manner that substantially reduces such cellular content of the placental product. Alternatively, maternal blood cells may be removed from the placenta by removing a substantial portion of one or more therapeutic components (e. G., Therapeutic agents including therapeutic cells (e. G., MSCs), angiogenic factors, antioxidants, Without the work being selectively removed or otherwise removed.

In one embodiment, removal of maternal blood cells comprises rinsing the amniotic membrane (e.g., with a buffer such as PBS) to remove total blood clots and any excess blood cells.

In one embodiment, the removal of maternal blood cells comprises treating the amniotic membrane using an anticoagulant (e. G. Citric acid dextrose solution).

In one embodiment, removal of maternal blood cells comprises rinsing the amniotic membrane (e.g., with a buffer such as PBS) to remove total blood clots and any excess blood cells, and administering an anticoagulant (e. G. Citrate dextrose solution) to treat the amniotic membrane.

In one embodiment, the chorion is maintained, and removal of maternal blood cells involves separating the chorion from the placenta by cutting around the placental skirt on the opposite side of the umbilical cord. The chorion on the umbilical side of the placenta is not removed due to angiogenesis on this side.

In one embodiment, the chorion is maintained, and removal of maternal blood cells is accomplished by separating the chorion from the placenta by cutting around the placental skirt on the opposite side of the umbilical cord, and the amniotic and chorionic membranes (e.g., using a buffer such as PBS ) Rinsing to remove total blood clots and any excess blood cells.

In one embodiment, the chorion is maintained, and removal of maternal blood cells involves separating the chorion from the placenta by cutting around the placenta skirt on the opposite side of the umbilical cord and treating with an anticoagulant (e.g., a citrate dextrose solution) .

In one embodiment, the chorion is maintained and removal of maternal blood cells is accomplished by separating the chorion from the placenta by cutting around the placental skirt on the opposite side of the umbilical cord, removing the chorionic membrane amniotic membrane (using, for example, a buffer such as PBS) Rinsing to remove total blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e. G. Citric acid dextrose solution).

TNFα  Emissive LPS  Selective depletion of immunogenicity as evidenced by substantial reduction in stimulation.

In one embodiment, the placental product is selectively depleted of immunogenicity as evidenced by a decrease in LPS-stimulated TNF-a release. In one embodiment, the placental product is selectively depleted of macrophages.

In one embodiment, TNF-a is depleted by the killing or elimination of macrophages.

In one embodiment, TNF- [alpha] is functionally depleted by treatment with PGE2 that inhibits TNF-a secretion.

In some embodiments, TNF- [alpha] is inhibited by at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

preservation

The placenta products of the present technology can be used fresh or can be preserved for a period of time. Surprisingly, cryopreservation in the art results in immunocompetent placental products.

In one embodiment, the placenta product is cryopreserved. The placenta product can be cryopreserved by incubation at a cryopreservation temperature in a cryopreservative solution (at about -20 캜 to about -196 캜, e.g., at -80 캜 5C).

Cryopreservation may include, for example, incubating the placental product at about 2 [deg.] C to about 8 [deg.] C for 30 to 60 minutes, and then incubating at -80 [deg.] C until use. The placenta product can then be thawed for use. Alternatively, the placenta product is cryopreserved in such a way that cell viability remains surprisingly well after the cryo-thaw cycle.

In one embodiment, the cryopreservation comprises storage in a cryopreserved solution comprising one or more cell-permeable cryopreservation agents, one or more non-cell-permeable cryopreservation agents, or a combination thereof. Optionally, the cryopreservation solution comprises one or more cell-permeable cryopreservation agents selected from DMSO, glycerol, glycols, propylene glycol, ethylene glycol, or combinations thereof. Optionally, the cryopreservation solution comprises one or more non-cell-permeable freeze-dried compositions selected from polyvinylpyrrolidone, hydroxyethyl starch, polysaccharides, monosaccharides, sugar alcohols, alginates, trehalose, raffinose, dextran, Preservative. Another example of a useful cryoprotectant is described in "Cryopreservation" (BioFiles, Volume 5, Number 4 Sigma-Aldrich (TM) Data Sheet).

In one embodiment, the cryopreservation solution comprises a cell-permeable cryopreservation agent, with the majority of cell-permeable cryopreservation agents being DMSO. Optionally, the cryopreservation solution does not contain a substantial amount of glycerol.

In one embodiment, the cryopreservation solution comprises DMSO. Optionally, the cryopreservation solution does not contain a large amount of glycerol. Optionally, the cryopreservation solution does not contain a substantial amount of glycerol.

In one embodiment, the cryopreservation solution comprises additional components such as albumin (e.g., HSA or BSA), electrolyte solution (e.g., Plasma-Lyte), saline solution or any combination thereof .

In some embodiments, the composition comprises a cryopreservation solution. The cryopreservation solution is added to a vessel containing the membrane fixture or composition. Preferably, a sufficient amount of the cryopreservation solution is added to the vessel to protect the membrane during the subsequent freezing step. Suitable cryopreservation solutions are known in the art. In one embodiment, the cryoprotectant comprises a cryopreservation solution comprising one or more cell-permeable cryopreservants, one or more non-cell permeable cryopreservants, or a combination thereof. Suitable cryopreservatives include, but are not limited to, DMSO, glycerol, glycols, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH) . In some embodiments, the cryopreservation solution is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, polysaccharide, monosaccharide, alginate, trehalose, raffinose, dextran, human serum albumin, phycol, lipoprotein, polyvinylpyrrolidone, One or more non-cell permeable cryopreservatives selected from hydroxyethyl starch, autologous plasma, or a combination thereof. Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4, Sigma-Aldrich (TM) Datasheet).

For example, a suitable cryopreservation solution may be stored in an amount of at least about 0.001% by volume to 100% by volume, suitably from about 2% by volume to about 20% by volume, preferably from about 5% by volume to about 10% Preservative. In some instances, the cryopreservation solution comprises at least about 2% cell-permeable cryopreservation agent. Additionally, the cryopreservation solution may comprise serum albumin or other suitable protein. In some embodiments, the cryopreservation solution comprises from about 1% to about 20% serum albumin or other suitable protein, alternatively from about 1% to about 10%. In one embodiment, the cryopreservation solution comprises between 1 vol% and about 15 vol% albumin and between about 5 vol% and about 20 vol% cryopreservation (e.g., about 10 vol%). Optionally, the cryoprotectant comprises DMSO (e. G., In large amounts). Serum albumin or other suitable protein is provided to stabilize the membrane during the freeze-thaw process and to maintain viability by reducing damage to the cells. The serum albumin may be human serum albumin or bovine serum albumin. The cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffered saline.

In one embodiment, the cryopreservation comprises placing placental membranes on nitrocellulose paper.

In one embodiment, placental membranes are cut into multiple sections prior to cryopreservation. Optionally, the section is placed on nitrocellulose paper prior to placing the placental membrane at about 2 [deg.] C to about 8 [deg.] C.

How to use

As discussed above, placental products, and in particular the cryopreserved membranes described herein (e. G., Amniotic membrane, chorionic membrane and / or villous amniotic membrane), are useful for the treatment of a number of beneficial therapeutic activities and effects, And the amount of extracellular matrix. In the methods disclosed herein, the membrane is provided with an environment capable of promoting the cell to provide an amount of therapeutic agent, survival cell and extracellular matrix, which not only provides a plurality of therapeutic factors but also provides the same or similar therapeutic advantages And can be applied and provided.

Accordingly, in one aspect, the disclosure provides a method of treating a wound in a subject comprising administering to the wound site a film comprising a cryopreserved amniotic membrane as described herein, wherein upon administration, the membrane comprises Extracellular matrix, and / or one or more therapeutic factors in an amount effective to promote one or more of:

(i) a decrease in the amount and / or activity of proinflammatory cytokines;

(ii) an increase in the amount and / or activity of anti-inflammatory cytokines;

(iii) reduction in the amount and / or activity of reactive oxygen species;

(iv) an increase in the amount and / or activity of an antioxidant;

(v) reducing the amount and / or activity of the protease;

(vi) increased cell proliferation;

(vii) increased angiogenesis; And / or

(viii) Increased cell migration.

In another aspect, the disclosure provides a method of accelerating wound healing comprising administering a membrane according to any of the aspects and embodiments described herein. In some embodiments, the administering step is effective to promote wound closure for up to 12 weeks after the initial administration. In some embodiments, the administering step is effective to promote wound closure for 5 to 6 weeks after the initial administration step. In some embodiments, the administering step is effective to promote a reduction in wound size by 50% or more by 28 days after the initial administration step. In an embodiment, the administering step is effective to improve wound closure rate by at least about 44% as compared to standard wound treatment.

Standard care for standard wound healing or wound treatment may refer to and include any treatment that does not incorporate a membrane or product as described herein. Suitable standard wound treatments are well known in the art and include, but are not limited to, application of dressings (e.g., gauze, bandages, barriers or bimetallically treated membranes containing undetectable or a few viable cells), necrosis, , Salve, ointment, and the like, and any combinations thereof. Standard wound treatments may include necrotic tissue removal with wound dressing or bandages and offloading devices.

In another embodiment, the disclosure provides a method of treating a subject for a wound that is refractory prior to a wound healing treatment, the method comprising administering a membrane to a wound site in accordance with any of the aspects and embodiments herein do. In some embodiments, the administering step is effective to promote wound closure for up to 12 weeks after the initial administration. In some embodiments, the administering step is effective to promote wound closure for 5 to 6 weeks after the initial administration step. In some embodiments, the administering step is effective to promote a reduction in wound size by 50% or more by 28 days after the initial administration step.

In another aspect, the disclosure provides a method of treating a chronic wound comprising administering to a wound site as disclosed herein, wherein the administering comprises administering to the surviving therapeutic cell an amount effective to promote healing of chronic wounds , An extracellular matrix and one or more therapeutic factors.

In an embodiment of the above aspects relating to wound healing, accelerated wound healing, and / or chronic wound healing, the wound may include a wound, a scar, a burn, a wound, a wound caused by a projectile, a skin wound, a skin wound, Selected from the group consisting of an acute wound, an external wound, an internal wound, a congenital wound, an ulcer, a pressure ulcer, a diabetic ulcer, a water wound, a wound caused by or in addition to a surgical procedure, a vein skin ulcer, and an avascular necrosis do.

In an embodiment of the above aspects of the wound healing method, the membrane comprises: (i) a reduction in the amount and / or activity of pro-inflammatory cytokines; (ii) an increase in the amount and / or activity of anti-inflammatory cytokines; (iii) reduction in the amount and / or activity of reactive oxygen species; (iv) an increase in the amount and / or activity of an antioxidant; (v) reducing the amount and / or activity of the protease; (vi) increased cell proliferation; (vii) increased angiogenesis; And / or (viii) an increase in cell migration, either directly or indirectly.

In another aspect, the disclosure provides a method of treating an inflammatory eye disease in a subject comprising administering to the subject a membrane as disclosed herein, wherein administration comprises administering a therapeutically effective amount of a surviving therapeutic cell , An extracellular matrix and one or more therapeutic factors. In an embodiment of this aspect, the method includes administration of the membrane using any technique that may be associated with promoting and / or reducing pain, and / or generally reducing inflammation of the eye tissue . For example, the membrane can be surgically administered via a surgical inlay or graft technique, via an onlay or patch technique, or through a combination of an inlay and an onlay technique. In general, the method can be used for the treatment of inflammation or healing which may result in eye surgery (e.g., photorefractive keratectomy (PRK)), eye trauma (e.g. lacerations, burns or abrasions) &Lt; / RTI &gt; Non-limiting examples of indications that include "inflammatory eye diseases &quot;, which are encompassed by the present methods include general restoration / reconstitution of the corneal or conjunctival surface (s), such as persistent epithelial defects, corneal ulcers; Corneal transplantation; Descemet's membrane prolapse; Corneal perforation; Defects after epithelial or subepithelial lesion or tumor incision (conjunctival tumor, conjunctival epithelial tumor, subepithelial lesion, target keratopathy, scar, conjunctival fold parallel to the edge of the eyelid); Acute chemical burn; Acute keratitis; Painful blister keratopathy; Partial or complete limbal stem cell deficiency (with stem cell transplantation); Acute Stevenson-Johnson syndrome; Penile adhesion; Reconstruction of the internal organs; Anorexia nervosa; Filtration repair; Scleroderma; And pterygia (see, for example, Meller, D., et al., Dtsch. Arztebl. Int., (2011); 108 (14): 243-248, incorporated herein by reference) .

In another aspect, the disclosure provides a method of promoting tissue repair and / or tissue regeneration in a subject comprising administering a membrane as disclosed herein to a subject, wherein administration comprises administering a tissue repair and / Cell, an extracellular matrix, and one or more therapeutic factors in an amount effective to stimulate the immune response. In some embodiments of this aspect, the method is used in conjunction with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery. In some embodiments, the tissue is a human tissue. In a further embodiment, the human tissue is cartilage, skin, ligament, tendon or bone. In this and various embodiments, the membrane can stimulate tissue regeneration directly or indirectly.

In another aspect, the present disclosure provides a method of modulating an inflammatory response comprising administering to a wound a membrane as disclosed herein, wherein the administration reduces the amount and / or activity of the proinflammatory cytokine and / Extracellular matrix, and / or one or more therapeutic factors in an amount effective to increase the amount and / or activity of the anti-inflammatory cytokine. In an embodiment, the pro-inflammatory cytokine is selected from TNF-a and IL-l [alpha], or a combination thereof. In one embodiment, the anti-inflammatory cytokine may be selected from IL-10 or PGE2 or a combination thereof.

In this aspect, the disclosure provides a method of modulating protease activity, comprising administering a membrane as described herein to a wound, wherein the administration comprises reducing the protease activity and / or reducing the amount and / or activity of the protease inhibitor Cell, an extracellular matrix, and / or one or more therapeutic factors in an amount effective to increase the amount of the compound of the invention. In an embodiment, the protease can be selected from the group consisting of a substrate metalloproteinase (MMP) and an elastase, or any combination thereof. In some embodiments, the at least one protease inhibitor comprises a tissue inhibitor of a substrate metalloproteinase (TIMP).

In a further aspect, the present disclosure provides a method of reducing the amount and / or activity of a reactive oxygen species (ROS) comprising administering a membrane as described herein to a wound, wherein administration comprises administering an amount of ROS and / Extracellular matrix, and / or one or more therapeutic factors in an amount effective to reduce the activity and / or increase the amount and / or activity of the antioxidant. In some embodiments, the antioxidant capacity provided by the membrane may be equivalent to a solution of ascorbic acid to about 250 mM. In some embodiments, the membrane can obtain cells from oxidant-induced apoptosis. In some embodiments, the membrane may reduce or prevent the cell from undergoing apoptosis after exposure to oxidants and / or after undergoing oxidative damage. In some aspects, the product is a cell that undergoes oxidant induced apoptosis by at least about 50%, alternatively at least about 60%, alternatively at least about 70%, alternatively at least about 80%, alternatively at least about 90% Can be reduced.

In another aspect, the disclosure provides a method of increasing angiogenesis, comprising administering a membrane as described herein to a wound, wherein the administration is effected in an amount effective to increase neovascularization, And / or one or more therapeutic factors. In some embodiments, the membrane provides an increase in the amount and / or activity of vascular epidermal growth factor (VEGF) or epidermal growth factor (EGF) or combinations thereof. In some embodiments, the membrane promotes angiogenesis.

In some embodiments, the membrane promotes angiogenesis. In some embodiments, the membrane may facilitate and / or improve the formation of a sutured tube or blood vessel within the tissue. The in vitro membrane facilitates the formation of a suture tube by HUVEC.

In another aspect, the disclosure provides a method of increasing cell migration comprising administering a membrane as described herein to a wound, wherein the administration comprises administering to the wound a cell, an extracellular matrix and / Or one or more therapeutic factors. In some embodiments, the membrane induces cell migration of cells selected from the group consisting of endothelial cells, fibroblasts and epithelial cells, and combinations thereof.

In another aspect, the disclosure provides a method of increasing cell proliferation comprising administering to a wound a membrane as described herein, wherein the administration is effected in an amount effective to increase cell proliferation, / RTI &gt; and / or one or more therapeutic factors.

In a further aspect, the disclosure provides a method of preventing or reducing scarring or build-up in a subject comprising administering to the subject a film as described herein, wherein the administration is to prevent or reduce scarring or build-up An effective amount of a survival therapeutic cell, an extracellular matrix and one or more therapeutic factors. In an embodiment of the method, the membrane may increase the amount or activity of IFN-2 alpha and / or TGF- beta 3 in an amount effective to prevent or reduce scar formation.

Modulate the inflammatory response, modulate protease activity, decrease the amount of reactive oxygen species, increase the amount of antioxidants, increase / promote neovascularization, increase cell migration, increase cell proliferation Or scar or build-up formation, the administration of the membrane may stimulate or induce the method directly or indirectly.

The placental product of the present technology can be used to treat any tissue injury. The therapeutic method can be provided, for example, by administering the placenta product of the present technique to a subject in need of treatment.

A typical method of administration of the technology is topical administration. The administering step of the technique may optionally involve the administration of an access to an internal tissue obtained by a surgical procedure.

The placental product may be administered autologously, allogeneically or heterologously.

In one embodiment, the placenta product is administered to a subject to treat a wound. Optionally, the wound is a laceration, abrasion, thermal burn or chemical burn, wound, hole or wound caused by a projectile. Optionally, the wound is a cutaneous wound, a skin wound, a chronic wound, an acute wound, an external wound, an internal wound, a congenital wound, an ulcer or a bedsore. Such wounds may be accidental or intentional, for example, wounds caused during or during surgical procedures. Optionally, the wound is sutured by surgery prior to administration.

In one embodiment, the placenta product is administered to a subject to treat a burn. Alternatively, the image may be an image of a first degree image, a second degree image (partial layer image), a third degree image (full layer image), an infection of a burn wound, an infection of an incision and a non-incision burn wound, Loss, or burn scratches.

In one embodiment, the placenta product is administered to a subject to treat an ulcer, e.g., a diabetic ulcer (e.g., a foot ulcer).

In one embodiment, the placenta product is administered by placing a placental product directly on the skin of the subject, for example on the stratum corneum, at the wound site, and thus the wound may be covered, for example, with an adhesive tape . Additionally or alternatively, the placenta product may be administered as an implant, for example, as a subcutaneous implant.

In one embodiment, the placenta product is administered to the epidermis to reduce wrinkles or other characteristics of aging. Such treatment is also usefully combined with so-called cosmetic surgery (e.g., laparoscopy, wrinkle resection, etc.).

In one embodiment, the placental product is used to accelerate healing associated with a dermis removal procedure or a dermis abrasion procedure (e.g., laser ablation, thermal ablation, electrical ablation, deep dermis removal, subepithelialization, To the epidermis.

Other pathologies that can be treated with placental products of the present technology include trauma wounds (e.g., civilian and military wounds), surgical scars and wounds, spinal fixation, spinal cord injury, avascular necrosis, reconstructive surgery, do.

In one embodiment, the placenta product of the technique is used in a tissue graft procedure. Alternatively, the placental product is applied to a portion of the graft and then attached to the biological substrate (e.g., to promote healing and / or adhesion to the substrate). As a non-limiting example, tissues such as skin, cartilage, ligament, tendon, osteoclast, cartilage, synovial membrane, fascia, mesentery and tendon can be used as tissue grafts.

In one embodiment, placental products are used in tendons or ligaments to promote healing of tendons or ligaments. Alternatively, the placental product is applied to a portion of the tendon or ligament attached to the bone. Surgery may be any tendon or ligament surgery including, for example, knee surgery, shoulder, leg surgery, arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, have. For example, placental products may be applied to the tendons or ligaments in implantation or reconstruction procedures to promote fixation of the tendons or ligaments to the bones.

DISCLOSURE OF THE INVENTION Throughout the present inventors, it has surprisingly been found that placental products of the present technology provide excellent treatment (e.g., healing, healing time and / or healing strength) for tendon and ligament surgery. Tendon and ligament surgery may entail fixation to tendon or ligament bones. Without being bound by theory, the inventors believe that the osteogenesis and / or cartilage potential of MSC in the present placenta product promotes healing process and healing strength of the tendon or ligament. The present inventors believe that the present placental product provides an alternative or additional treatment for a peletal-based treatment. For example, useful osteoblast-based therapies are described in Chen et al. 2003; 19 (3): 290-6). Chen et al. (2003) reported that the tendon-bone healing in a bone tunnel was superior to that of the tendon graft with periosteum. ("Enveloping of periosteum on the hamstring tendon graft in anterior cruciate ligament reconstruction"; Arthroscopy 2002 May-Jun; 18 (5): 27E), Chang et al. (&Quot; 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) .

As a non-limiting example of a tendon or ligament surgical procedure, tendons are sutured and / or wrapped or encased in placental membranes, and tendons attached to the bones. Optionally, the tendon is placed into the bone tunnel prior to attachment to the bone.

In one embodiment, tendon or ligament surgery is a graft procedure, and the placenta product is applied to the graft. Alternatively, the graft is a homograft, a xenograft, or a graft.

In one embodiment, tendon or ligament surgery is a repair of torn ligaments or tendons, and placental products are applied to torn ligaments or tendons.

Non-limiting examples of tendons to which placental products may be applied include femoris tendon, hamstring tendon, biceps tendon, Achilles tendon, extensor, and rotator cuff tendon.

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

In one embodiment, the placenta product of the present technique is used as an anti-adhesion wound barrier, wherein the placenta product is applied to the wound site to reduce, for example, fibrosis (e.g., post-surgical fibrosis).

Non-limiting examples of wound sites to which placental products may be applied include surgery induced or induced by neovascularization, including spinal, choledochotomy, knee, shoulder or delivery, trauma related injuries or injuries, Stimulation, brain / neurological procedures, burns and wound healing, and ophthalmic procedures. For example, optionally, the wound site is associated with the operation of the vertebrae, and the substrate side of the placenta product is applied to the dura (e.g., the substrate side in contact with the dura). Directions for such procedures, including selection of wound sites and / or methods, can be found in, for example, WO 2009/132186 and US patent application no. 2010/0098743, incorporated herein by reference.

The placental product of the present technique may optionally be used to reduce adhesion of the wound or fibrosis. Postoperative fibrosis is a natural consequence of all surgical wound healing. As an example, postoperative epidural adhesions result in tetraling, traction and compression of the spinal dural sac and neuromuscular muscles that cause recurrence of the sensory hypersensitivity typical of a few months after surgery. Repeated surgeries for removal of scar tissue are associated with poor outcomes and increased risk of injury due to the difficulty of identifying the nerve structures surrounded by scar tissue. Thus, empirical and clinical studies have focused primarily on preventing scar tissue adhesion to the dural and nerve roots. Spinal adhesion was implicated as a major contributing factor in the failure of spinal surgery. Fibrosis Scar tissue causes compression and tethering of the nerve roots, which may be associated with recurrent pain and disability.

Without being bound by theory, the present inventors have found, at least in part, that the placental product inhibits a reduced number of immune cells as well as an increased number of cellular factors (e.g., TGF-β3, HGF, VGF, EGF, HE, Etc.), it is believed that the placental product taught herein is useful for reducing adhesion or fibrosis of the wound. One advantage of the wound dressings and procedures of the present technology is that they provide anti-adhesion barriers that can be used to prevent adhesion after surgery and especially after back surgery.

In the preceding paragraphs, the use of the singular may include a plurality, except where specifically indicated. As used herein, the word "one or more" means &quot; one or more &quot; In addition, where aspects of the present technology are described in connection with a list of alternatives, the techniques include any individual member or subgroup of alternatives and a list of any combination of one or more of the foregoing.

The disclosures of all patents and publications, including those disclosed in the open patent applications, are incorporated herein by reference to the same extent as if each patent and publication was specifically and individually indicated to be incorporated by reference.

It is to be understood that the scope of the present technology is not limited to the specific embodiments described above. The present technology may be practiced other than as specifically described and still within the scope of the following claims.

Likewise, the following examples are presented to more fully illustrate the technique. They should, however, not be interpreted in any way as being limited to the broad scope of the techniques disclosed herein.

The techniques described herein and their advantages will be more readily appreciated with reference to the following examples. These embodiments are provided to describe specific embodiments of the technology. By way of providing these specific embodiments, they are not intended to limit the scope or the extent of the present technique. It will be understood by those skilled in the art that the entire scope of the presently described technology includes the subject matter defined by the claims appended hereto and any alterations, variations or equivalents of such claims.

Example

Other features and embodiments of the present technology will be apparent from the following examples, which are provided by way of illustration of the present technology, rather than limiting its intended scope.

Manufacturing process

Example 1 Exemplary Manufacturing Process of Amniotic Membrane Product

In one embodiment, and as discussed herein, this disclosure relates to a method of making a placental product (or, alternatively, the "membrane" in the following embodiments) comprising amniotic membrane from placenta after parturition . One such method includes the following:

a. Removal of umbilical cord close to the placenta surface,

b. Dull amniotic membrane detachment of placenta skirt,

c. The placenta is inverted to completely remove the amniotic membrane,

d. The amniotic membrane was rinsed in PBS to remove red blood cells,

e. The amniotic membrane was rinsed once with 11% ACD-A solution to help remove red blood cells,

f. The amniotic membrane was rinsed with PBS to remove the ACD-A solution,

g. Any remaining blood was removed from the amniotic membrane using PBS,

h. Gently remove any other ingredients that are not part of the amniotic membrane epithelium and the substrate layer,

i. Place the amniotic membrane in PBS and set aside,

j. The amniotic membrane is placed in a bottle containing the antibiotic solution and incubated at 37 ° C ± 2 ° C for 24-28 hours,

k. The bottle is removed from the incubator, the membrane is rinsed with PBS to remove the antibiotic solution,

l. Amniotic membrane (epithelial side up) is placed on the reinforced nitrocellulose paper, cut to the right size,

m. Packed in an FP-90 frozen bag and heat-sealed,

n. A 50 mL cryopreservation solution was added to the bag through a syringe and the syringe was used to remove any air trapped in the bag,

o. Tube sealing of the solution line of the FP-90 bag,

p. The bag was poured into a secondary bag, heat sealed,

q. Placing the unit into a packaging carton,

r. Refrigerate for 30 to 60 minutes at 2 to 8 캜 and freeze at -80 캜 5 캜 in a styrofoam container.

Example  2 &lt; / RTI &gt; Preparation of Placenta Product Containing Chorionic Film

One way to produce placental products containing chorionic villi and optionally amniotic membrane from placenta after delivery:

a. Removal of umbilical cord close to the placenta surface,

b. Dull amniotic membrane detachment of placenta skirt,

c. The placenta is inverted to completely remove the amniotic membrane,

d. Removing the chorion by cutting around the placenta skirt,

e. Both membranes were rinsed in PBS to remove red blood cells,

f. Both membranes were rinsed once with 11% ACD-A solution to help remove red blood cells,

g. Both membranes were rinsed with PBS to remove the ACD-A solution,

h. Treating the chorions in 200 ml 0.5% dispase solution at 37 ° C ± 2 ° C for 30-45 minutes,

i. When the dispase treatment is complete, the chorion is rinsed with PBS to remove the dispase solution,

j. Smoothly remove the trophoblast from the chorion,

k. The chorion is placed in a bottle containing the antibiotic solution and incubated at 37 ° C ± 2 ° C for 24-28 hours,

l. The bottle was removed from the incubator, the chorion was rinsed with PBS to remove the antibiotic solution,

m. The chorion is placed on cellulose paper with reinforced nitrocellulose, cut to the right size,

n. Each piece was placed in an FP-90 freeze bag and heat sealed,

o. A 50 mL cryopreservation solution was added to the bag through a syringe and the syringe was used to remove any air trapped in the bag,

p. Tube sealing of the solution line of the FP-90 bag,

q. The bag was put into a secondary bag, and heat-sealed,

r. Placing the unit into a packaging carton,

p. Refrigerate at 2-8 [deg.] C for 30-60 minutes,

t. Frozen in Styrofoam container at -80 ° C ± 5 ° C.

Example 3: Exemplary preparation of villous amniotic membrane product

The present disclosure provides a method of making a placental product comprising amniotic membrane and chorionic membrane from placenta postpartum, comprising:

a. Removal of umbilical cord close to the placenta surface,

b. Dull amniotic membrane detachment of placenta skirt,

c. Overturning the placenta,

d. Cutting around the placenta skirt removes the chorion and attached amniotic membrane (villous amniotic membrane)

e. Both membranes were rinsed in PBS to remove red blood cells,

f. Both membranes were rinsed once with 11% ACD-A solution to help remove red blood cells,

g. Both membranes were rinsed with PBS to remove the ACD-A solution,

h. The membrane is placed in a bottle containing the antibiotic solution and incubated at 37 ° C ± 2 ° C for 24-28 hours,

i. The bottle is removed from the incubator and rinsed with PBS to remove the antibiotic solution,

j. Smoothly remove the trophoblast from the chorion,

k. The vitreous membrane is placed on cellulose paper with reinforced nitrocellulose, cut to the right size,

l. Each piece was placed in an FP-90 freeze bag and heat sealed,

m. A 50 mL cryopreservation solution was added to the bag through a syringe and the syringe was used to remove any air trapped in the bag,

n. Tube sealing of the solution line of the FP-90 bag,

o. The bag was put into a secondary bag, and heat-sealed,

p. Placing the unit into a packaging carton,

q. Refrigerate at 2-8 [deg.] C for 30-60 minutes,

r. Frozen in Styrofoam container at -80 ° C ± 5 ° C.

Example  4 Example of Placental Product Manufacturing Method

A further detailed description of one method for the preparation of placental product membranes containing amniotic membrane according to the presently disclosed manufacturing process is provided below.

The placenta was processed in a biological safety cabinet. First, the umbilical cord was removed, and the amniotic membrane was detached from the underlying chorion using blunt dissection. The membranes are rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, NY) to remove lumps and any excess blood cells. The membrane was then treated with 11% anticoagulant citrate dextrose solution (USP) formulation A (ACD-A) (Bacterium Healthcare, Deerfield, IL) in saline (Baxter Healthcare Corp, Deerfield, Ill. &Lt; / RTI &gt; corporation) to remove the remaining blood cells.

The amniotic membrane substrate layer was cleaned by gently scraping any other components that were not part of the epithelium and substrate layers.

Amniotic membrane was then cultured in Dulbecco's Modified Eagle's Medium (DMEM) at 37 ° C ± 2 ° C for 24-28 hours with gentamicin sulfate (50 μg / ml) (Abraxis Pharmaceuticals, Shamburg, Ill. (Sigma Aldrich, St. Louis, MO)), vancomycin HCl (50 μg / mL) (Hospira, Lake Forest, Ill.) And amphotericin B 0.0 &gt; mL &lt; / RTI &gt; of the solution. After the incubation period, the membrane was washed with PBS to remove any residual antibiotic solution.

The membrane was fixed on an Optitran BA-S 85 fortified nitrocellulose paper (Whatman, Germany Dassel) and packed in a FP-90 freeze bag (Charter, Inc., Winston-Salem, North Carolina) Medical Ltd.)). The substrate side of amniotic membrane was fixed against nitrocellulose paper. Once the membrane unit was placed in an FP-90 frozen bag and the frozen bag was heat-sealed, 10% dimethyl sulfoxide (DMSO) (Bioniche Teo Inverin Co., Galway, Ireland) and 5 50 mL of cryopreserved solution containing 10% human serum albumin (HSA) (Bauter, Westlake Village, CA) was added via the center tubing line. After any excess air was removed, the tubing line was subsequently sealed.

The FP-90 frozen bag was placed into a manger bag (10 inches by 6 inches) (Mangar Industries, New Britain, Pa.) And then heat sealed. The mugger bag was placed in a packaging carton (10.5 inches x 6.5 inches x 0.6 inches) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8 캜 for 30-60 minutes before freezing at -80 캜 5 캜 in Styrofoam containers.

Example 4.1 Thawing time for membranes fixed on nitrocellulose paper.

The cryopreserved membranes described herein can exhibit properties that thaw faster than other cryopreserved products. Fast thawing profiles enable the films described herein to be used efficiently and effectively for demanding applications and applications. One thawing protocol is discussed below.

Two samples were taken from two lots of placenta tissue packaged on nitrocellulose paper taken from a cryogenic freezer (-80 占 폚).

A frozen bag containing placenta tissue was placed in the thawing chamber filled with water at room temperature.

The thawing time determined when no observable ice crystals remained was recorded.

result

Thawing time for placental membrane products frozen with 50 mL cryopreservation solution. Donor Thawing time (min) A 24 B 28 Average 26

While thawing is complete, all ice crystals must disappear. Alternatively, the weight of the ice may cause the membrane to tear or cause the membrane to fall off the cellulose paper to cause self-folding when thawing is complete. The results are shown in Table 1 above and demonstrate that the average thawing time is 26 minutes and thus facilitates "on demand" application and application to the cryopreserved membranes described herein.

Example 5. Exemplary preparation of chorioamnion product

One method of preparing placental products, including amniotic membrane products and chorion, according to the presently disclosed manufacturing procedure is as follows:

The placenta was processed in a biological safety cabinet. First, the umbilical cord was removed, and the chorion attached to the amniotic membrane was cut from the placenta skirt. Both membranes are rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, NY) to remove lumps and any excess blood cells. The membrane was then treated with 11% anticoagulant citrate dextrose solution (USP) formulation A (ACD-A) (Bacterium Healthcare, Deerfield, IL) in saline (Baxter Healthcare Corp, Deerfield, Ill. &Lt; / RTI &gt; corporation) to remove the remaining blood cells.

The chorion amniotic membrane was then treated with gentamicin sulfate (50 μg / mL) (Abraxis Pharmaceutical Products, Inc., Schaumburg, IL), vancomycin HCl (Sigma Aldrich, St. Louis, MO), and Amphotericin B (2.5 占 퐂 / mL) (Sigma Aldrich, St. Louis, MO) Respectively. After the incubation period, the membrane was washed with PBS to remove any residual antibiotic solution. The trophoblastic layer of the chorion was gently removed using dull exfoliation.

The membrane was fixed on an Optitran BA-S 85 fortified nitrocellulose paper (Whatman, Germany Dassel) and packed in a FP-90 freeze bag (Charter, Inc., Winston-Salem, North Carolina) Medical Ltd.)). Once the membrane unit was placed in an FP-90 frozen bag and the frozen bag was heat-sealed, 10% dimethyl sulfoxide (DMSO) (Bioniche Teo Inverin Co., Galway, Ireland) and 5 50 mL of cryopreserved solution containing 10% human serum albumin (HSA) (Bauter, Westlake Village, CA) was added via the center tubing line. After any excess air was removed, the tubing line was subsequently sealed.

The FP-90 frozen bag was placed into a manger bag (10 inches by 6 inches) (Mangar Industries, New Britain, Pa.) And then heat sealed. The mugger bag was placed in a packaging carton (10.5 inches x 6.5 inches x 0.6 inches) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8 캜 for 30-60 minutes before freezing at -80 캜 5 캜 in Styrofoam containers.

Example 6 Quantitative evaluation of cell number and cell viability after enzymatic degradation of placental membrane

The amniotic membrane and chorion and the placental membrane products (from above) were evaluated for overall cell viability and cell viability throughout the course. These analyzes were performed on new placental tissue (before antibiotic treatment), placental tissue after antibiotic treatment, and product units after thawing. Cells were isolated from placental membranes using enzyme digestion. For cryopreserved product units, FP-90 frozen bags were first removed from the packaging cartons and machetes. The FP-90 frozen bag was then thawed for 2 to 3 minutes in a water bath at room temperature. Initial experiments involved the use of a 37 ° C ± 2 ° C water bath. After thawing, the placental membrane was removed from the FP-90 frozen bag and placed in a reservoir containing saline (Bartter Healthcare Corporation, Deerfield, Ill.) For a minimum of 1 minute and a maximum of 60 minutes. Each membrane was separated from the reinforced nitrocellulose paper before disassembly.

Amniotic membrane was resolved on a rocker at 37 ° C ± 2 ° C for 20 to 40 minutes with 40 mL of 0.75% collagenase (Worthington Biochemical Corp., Lakewood, NJ) solution. After collagenase digestion, the samples were centrifuged at 2000 rpm for 10 minutes. After removing the supernatant, 30 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, MD) was added and then incubated for an additional 5-15 minutes on the rocker at 37 ° C ± 2 ° C. Trypsin was warmed to 37 ° C ± 2 ° C in a water bath before use. After trypsin digestion, the supernatant was filtered through a 100 [mu] m cell strainer nylon filter to remove any debris. 15 ml DMEM was passed through the strainer into the same centrifuge tube. After centrifugation at 2000 rpm for 10 minutes, the supernatant was removed. The cell pellet was reconstituted using a volume of DMEM proportional to the pellet size and then 20 μL of the resuspended cell suspension was mixed with 80 μL trypsin blue (Sigma Aldrich, St. Louis, MO) for digestion. Cell count samples were placed in the hemocytometer and assessed using a microscope.

The chorion was detached on a rocker at 37 ° C ± 2 ° C for 20 to 40 minutes using 25 mL of 0.75% collagenase solution. After collagenase digestion, the supernatant was filtered through a 100 [mu] m cell strainer nylon filter to remove any debris. After centrifugation at 2000 rpm for 10 minutes, the supernatant was removed. The cell pellet was reconstituted using a volume of DMEM proportional to the pellet size and then 20 μL of the resuspended cell suspension was mixed with 80 μL of trypsin blue for digestion. Cell count samples were placed in the hemocytometer and assessed using a microscope.

Placental membranes were analyzed before any processing to determine the initial properties of the membranes.

Table 2 contains the mean cell and cell viability values per square centimeter for amniotic membrane and chorion from 32 placental lots.

On average, there were 91,381 viable cells per square centimeter of amniotic membrane and corresponding mean cell viability was 84.5%. For the chorion, there were 51,614 viable cells per cm &lt; 2 &gt;, corresponding cell viability was 86.0%.

These data may include the number of cells useful by certain embodiments of the present technology; For example, a placenta product comprising amniotic membrane containing from about 10,000 to about 360,000 cells / cm2 is illustrated. Since the amniotic membrane is composed of epithelial cells and stromal cells, experiments were conducted to determine the ratio of epithelial to stromal cells. Amniotic membranes from three placenta litters were analyzed. First, a 5 cm x 5 cm piece of amniotic membrane was resolved in a water bath for 30 minutes at 37 ° C ± 2 ° C using approximately 25 mL of 0.05% Trypsin-EDTA (Lonza, Walkersville, MD). After the incubation step, epithelial cells were removed by gentle scraping of cells from the membrane. After rinsing with PBS (Gibco Invitrogen, Grand Island, NY), the membrane was subsequently degraded in the same manner as the chorion (described above). In addition, another amorphous 5 cm x 5 cm piece of amniotic membrane was digested using standard procedures (described above) to determine the total number of cells. Subsequently, the percentage of stromal cells was determined by dividing the number of cells from the amniotic membrane from the epithelial cells by the number of cells from the defect-free membrane.

The results indicate that 19% of total cells are stromal cells. Thus, approximately 17,362 stromal cells are present in the amniotic membrane with approximately 74,019 epithelial cells. These data indicate that there are approximately three times more stromal cells in the chorioamnion than in the amniotic membrane. This ratio is consistent with certain embodiments of the present technology that provide a placenta product comprising chorionic villi and amniotic membrane, wherein the choroidal membrane comprises about 2 to 4 times more stromal cells than amniotic membrane.

Survival cells and cell viability per ㎠ were evaluated after the antibiotic treatment step. The treated cell recovery was calculated by comparing the number of viable cells before and after antibiotic treatment (as previously described in the technique described herein). Table 3 provides the results from these analyzes.

Example 7 Development of Placental Product Freezing Preservation Procedure

Cryopreservation is a method of providing a source of tissue and living cells. The main purpose of cryopreservation is to minimize damage to biological material during cryogenic refrigeration and storage. Although general cryopreservation rules are applicable to all cells, tissues and organs, certain cryopreservation solutions and procedures must be developed for each type of biological material. This application discloses a cryopreservation procedure for placental membrane products that can selectively deplete immunogenic cells from placental membranes and preserve the viability of other beneficial cells that are the primary source of factors for promoting healing.

During development of the cryopreservation method for placenta membranes, the present inventors evaluated important parameters of cryopreservation, including the volume of the cryopreservation solution, the effect of pre-cryoprotective state, and the cooling rate for the cryopreservation procedure.

In the absence of immunosuppression, the acceptance of tissue allografts will depend on the number of satellite immune cells present in the tissue. Cryopreservation is an approach that can be used to reduce tissue immunogenicity. This approach is based on the differential susceptibility of different cell types to cryopreservation in the presence of DMSO; Leukocytes are sensitive to fast cooling rates. The freezing rate of 1 [deg.] C / min is considered optimal for cells and tissues containing immune cells. Fast freezing rates, such as 60-100 ° C / min, remove immune cells. However, this type of procedure is detrimental to other tissue cells desirable for conservation in accordance with the present technique. The cryopreservation procedure developed uses a cryopreservation solution containing 10% DMSO, which is an important component that protects cells from destruction when water forms crystals at low temperatures. The second stage of cryopreservation was the complete equilibrium state of the placenta membranes in the cryopreservation solution, which was achieved by immersion of the membranes in the cryopreservation solution at 4 DEG C for 30 to 60 minutes. This step allows DMSO to penetrate the placental tissue. Some data indicate that pre-freeze tissue equilibrium can affect the survival of lymphocytes (e.g., Taylor & Bank, Cryobiology , 1988, 25: 1); However, this data indicates that even after tissue equilibrium, a substantial number of macrophages survive.

The placental membrane equilibrium in the DMSO-containing solution at about 2 ° C to about 8 ° C unexpectedly prevented placental membrane equilibrium in the presence of macrophages for freezing such that these types of cells could not withstand freezing even at a rate of about 1 ° C / Lt; RTI ID = 0.0 &gt; immune &lt; / RTI &gt;

Temperature mapping experiments were performed to analyze the temperature profiles of potentially cryopreservation conditions of the film product. These results are shown in Fig. Eight (8) FP-90 frozen bags were filled with 20 mL or 50 mL of cryopreserved solution, and then the temperature probe was placed inside each freezing bag. The first set of parameters (conditions 1 to 4 of Figures 1a to 1d, respectively) was followed by a refrigerated (2-8 ° C) step for 30 minutes before freezing (-80 ° C ± 5 ° C). In addition, the analysis involved freeze bag freezing in styrofoam containers or in freezer storage. The second set of parameters (conditions 5 to 8 of FIGS. 1E-1H, respectively) involved direct refrigeration (-80 ° C ± 5 ° C) of the freeze bag in the styrofoam container or in the freezer storage. The results indicated that condition 6 and condition 2 showed the most gradual temperature decrease. Progressive temperature reduction is typically desired to preserve cell viability. The difference between condition 6 and condition 2 is that condition 2 involved a 30 min refrigeration step. Therefore, in the case of latent heat progression during freezing, the temperature was further tested to decrease to -4 캜 from the beginning of the freezing. For condition 6, the cooling rate during this period was approximately -1 DEG C / min. The cooling rate for Condition 2 was approximately -0.4 ° C / min during the same time frame. Thus, condition 2 was chosen for incorporation into a non-limiting cryopreservation process, since slower cooling rates are generally desired to maintain optimal cell viability.

Figure 2a shows the effect of cryopreserved solution volume on cell recovery for treated (cryopreserved) amniotic membrane. Figure 2b shows the effect of a cryopreserved solution on cell viability for amniotic membrane.

The treatment (cryopreserved) cell count was calculated by comparing to viable cell count before and after cryopreservation (as described herein before). The intention was to identify cryopreservation conditions that provide the maximum cell count. However, the analysis used (except trypsin blue) requires taking tissue samples from different parts of the placenta, and it is impossible to obtain accurate cell recovery measurements, since each tissue sample contains a different number of cells. This accounts for a large range of treated cell counts (10% to 410%) encountered by the present inventors. On the other hand, cell viability was calculated by comparing the number of living cells to the total number of cells in the same piece of tissue. Thus, cell viability was used to optimize the parameters of the cryopreservation procedure.

As shown in FIG. 2B, a 50 mL volume of the cryopreservation solution volume provided the same cell count compared to 10 mL and 20 mL for the 5x5 placental membrane. These data indicate that a 10 mL cryopreservation solution volume is sufficient to provide a placental product with a cell viability of greater than 70% according to the present technique. The same results were obtained for the chorioamnion as can be seen from Figs. 3A and 3B.

Experiments were conducted to evaluate whether the cryopreservation method could be applied to the preservation of defect - free villous amniotic membrane. Chorionic amniotic membrane was excised from new placenta and incubated overnight in antibiotics. Subsequently, the membrane was cut into 2 cm x 2 cm pieces after removing the nutrient layer. Both membranes were maintained throughout the entire process to remain intact. New and cryopreserved placental membranes were prepared and cell viability was measured using MTT assay (Biotium 30006), a colorimetric assay for measuring cell viability. The analytical mechanism is based on the fact that the metabolic reduction of the soluble tetrazolium salt to the blue formazan precipitate depends on the presence of viable cells with defect-free mitochondrial function. Samples were incubated in the MTT assay medium for 3 to 4 hours. At the end of the conversion period, samples were extracted in DMSO for 1 hour at 37 占 폚. At the end of the extraction period, 0.2 mL of each extract was transferred to the wells of a 96-well plate. Absorbency proportional to cell viability was absorbed on a plate reader (Spectramax 340PC, Molecular Devices) at 570 nm using DMSO extraction buffer as a blank. Figure 4 shows the MTT values obtained from fresh and thawed cryopreserved samples. As shown by the data, cell viability did not show a significant difference between fresh cryopreserved villus amniotic membrane samples and thawed freeze-preserved villus amniotic membrane samples.

Experiments were conducted to evaluate different potential freezing conditions that maximize cell recovery after cryopreservation treatment. Figures 5A (amniotic membrane) and Figure 5B (chorion) show these results showing the effect of refrigeration time and refrigeration parameters on treatment (cryopreserved) cell recovery on chorion. Three conditions were analyzed. These conditions were related to temperature mapping studies. The first condition involved a step of freezing the product unit directly during storage in the freezer (-80 ° C ± 5 ° C). The second condition also included direct freezing, but the product unit is placed in a styrofoam container in the freezer. The third condition included a refrigeration (about 2 캜 to about 78 캜) period 30 minutes before the refrigeration step. For the amniotic membrane, three placenta lots were evaluated. Two (2) placental lots were analyzed for chorioamnion. The results indicated that the third condition was optimal for both membrane types.

The effect of refrigeration time and freezing parameters on amniotic membrane cell viability was also tested. When the absorbance was proportional to cell viability, cell viability was measured using the MTT assay described above. Four temperature equilibration times were tested: direct freezing (0 hr), equilibrium in a refrigerator (4 ° C) for 1 hour or 4 hours and equilibrium at room temperature for 4 hours. For each condition, it was also tested to freeze the product either in the freezer storage or directly in the styrofoam box. Figure 6 shows the results of the study and shows that the highest cell viability is obtained when the amniotic membrane is stored at 4 DEG C for up to 1 hour and then frozen in the styrofoam box. This condition was chosen for further development as it provided the most extensive cell recovery and cell viability.

All cryopreservation parameters evaluated for amniotic membrane and chorioamnion are summarized in Tables 4 and 5.

Evaluation of cell viability from these experiments led to the selection of the final parameters for the manufacturing process. In addition, all the mean cell viability values were above 70%.

These data are consistent with certain embodiments of the present technology to provide a placental product membrane comprising amniotic membrane containing from about 40,000 to about 90,000 or about 260,000 cells / cm2.

Example  8. Stability of cryopreserved amniotic membrane

One unique aspect of the technique is that the cryopreservation method can preserve cell viability over a prolonged period of time. To test whether cell viability is maintained after two years of storage, three lots of amniotic membrane were prepared using final preparation and cryopreservation treatment. Cell viability was determined using the quantitative method described in Example 6 at 0 hours (1 week storage), 12 months, 24 months and 25 months after storage. Table 6 demonstrates that after prolonged storage and subsequent thawing, cell viability remains above 70% for all lots at all time points.

Example  9 Cells by Tissue Dyeing Survival  Quantitative evaluation

Amniotic membrane and chorion were stained with Live / DEAD (TM) Viability / Cytotoxicity Kit (Molecular Probes Inc., Eugene, Oreg.) To determine cell viability Were quantitatively evaluated. Dyeing was performed according to the manufacturer's protocol. A film segment of approximately 0.5 cm x 0.5 cm was used. Evaluation of dye films was performed using a fluorescence microscope. Strong and uniform green fluorescence showed the presence of living cells, and bright red light showed the presence of apoptotic cells. Images of fresh amniotic membrane and chorion, as well as cryopreserved amniotic membrane and chorionic membrane, proved that the manufacturing process did not alter the phenotypic characteristics of the membrane after thawing.

FIG. 7 is a schematic diagram showing the results of a new amniotic membrane epithelium (A), a cryopreserved amniotic membrane epithelium (B), a new amniotic membrane substrate layer (C), a cryopreserved amniotic membrane substrate layer (D) And a representative image of the chorion (F). Living cells are green, and dead cells are red. These images demonstrate that the manufacturing process did not alter the phenotypic characteristics of the membrane and the proportion of surviving cell types (epithelial and stromal cells) in the membrane after thawing.

Example 10 An exemplary preparation procedure removes immunogenic elements from placental membranes

One unique feature of the human amnion and chorion is the absence of fetal blood vessels that prevent migration of white blood cells from the fetal circulation. On the fetal side, macrophages present in the villous amniotic membrane mesoderm layer represent only a population of immune cells. Thus, fetal macrophages present in the amniotic membrane and chorion are the major sources of tissue immunogenicity. However, the number of macrophages in the amnion is significantly lower than that of chorionic villi (Magatti et al, Stem Cells, 2008, 26: 182), because of the low immunogenicity of amniotic membrane and its ability to cross HLA without matching between donor and recipient (Akle et al, Lancet, 1981, 8254: 1003; Ucakhan et al., Cornea, 2002, 21: 169). In contrast, chorioamnion is considered immunogenicity. In studies using amniotic membrane with chorionic villi for reconstruction of conjunctival defects, the success rate was low (De Roth Arch Ophthalmol, 1940, 23: 522). Without being bound by theory, the inventors believe that removal of parental and CD14 + fetal macrophages from placental membranes among other immunogenic cell types prevents the activation of in vitro lymphocytes. The removal of the parental supernatant can be achieved by direct washing, whereas the cryopreservation treatment described in this technique removes CD14 + cells.

The immunogenicity test was used to characterize placental product membranes as safe clinical treatments. The immunogenicity of the placental products was tested at different manufacturing steps using two biomolecular analyzes (mixed lymphocyte reaction (MLR) and lipopolysaccharide (LPS) -induced tumor necrosis factor (TNF) -α secretion).

Example 10.1 Mixed Lymphocyte Reaction (MLR)

MLR is a type of in vitro assay widely used to test cell and tissue immunogenicity. The assay is performed from an individual that recognizes homologous human leukocyte antigen (HLA) and other antigenic molecules expressed on the surface of allogeneic cells and tissues (stimulators) derived from other individuals when mixed together in the wells of an experimental tissue culture plate Based on the ability of the derived immune cell (the responder). The response of immune cells to stimulation by allogeneic cells and tissues can be measured by a variety of methods including, but not limited to, the administration of certain cytokines (e.g., secretion of interleukins (IL-2), expression of specific receptors Can be measured using cell proliferation, all of which are characteristic of activated immune cells.

Placental tissue samples representing the different steps of the presently disclosed manufacturing process were used for immunogenicity testing. These samples included amniotic membrane with chorionic villus (ACT) as a starting material and isolated chorionic villus (CT), chorionic membrane (CM), trophoblast (T), amniotic membrane (AM), amniotic membrane and chorionic membrane (A / Both were purified and cryopreserved (end product) tissue was tested.

For MLR analysis, cells from placental tissue were isolated using 280 U / mL type II collagenase (Worthington, catalog number 4202). Tissue was treated with enzyme at 37 ° C ± 2 ° C for 60 to 90 minutes and then the resulting cell suspension was filtered through a 100 μm filter to remove tissue debris. The single cell suspension was then centrifuged at 2000 rpm for 10 minutes using Beckman, TJ-6 and washed twice with DPBS. The supernatant was discarded after each wash and the cells were resuspended in 2 mL of DMEM (Invitrogen, Cat. # 11885) and then resuspended in the presence of Trypan blue dye (Invitrogen, catalog # 15250-061) Cells were enumerated and evaluated for cell number and cell viability. Placenta derived cells were mixed with homologous hPBMC at a ratio of 1: 5 in a 24-well culture plate in DMEM supplemented with 5% fetal bovine serum (FBS) and then incubated at 37 ° C ± 2 ° C with 5% CO ₂ , 95% Lt; / RTI &gt; for 4 days. Human peripheral blood mononuclear cells (hPBMC) were used alone as negative controls and a mixture of two sets of hPBMCs derived from two different donors was used as a positive MLR control. After 4 days of incubation the cells were harvested from the wells and lysed with lysis buffer supplemented with protease inhibitor cocktail (Roche, Cat # 11836153001) (Sigma, Cat # C2978) 2R [alpha] was measured in cell lysates using the IL-2R [alpha] ELISA kit (R & D Systems, catalog number SR2A00) according to the manufacturer's protocol.

IL-2R [alpha] levels are measures of activation of T cells that respond to immunogenic molecules expressed by allogeneic cells. The results presented in Figures 8 and 9 demonstrate a method of producing placental membranes resulting in low immunogenicity of the final product.

Figure 8 demonstrates that the manufacturing process continuously reduces the immunogenicity of placental products. Samples representing the different steps of the manufacturing process were co-incubated with hPBMC for 4 days. IL-2R [alpha] was measured in cell lysates as a marker of T-cell activation. Negative controls indicate basal levels of immune cell activation: PBMCs derived from one donor were cultured alone. Positive Control: A mixture of PBMCs derived from two different donors.

Figure 9 demonstrates that selective depletion of immunogenicity results from the present cryopreservation process that produces the placenta product, as evidenced by a significant reduction in immunogenicity upon cryopreservation.

Example 10.2 LPS-Induced TNF-a Secretion by Placental Membrane Cells

As described herein, fetal macrophages present in the amniotic membrane and chorion are the major sources of tissue immunogenicity. Without being bound by theory, the inventors believe that CD14 + fetal macrophages from placental membranes prevent lymphocyte activation and reduce inflammatory cytokine secretion and tissue immunogenicity levels. Macrophages in the fetal placenta respond to bacterial LPS by secretion of inflammatory cytokines such as TNF-α. Thus, secretion of TNF-a responsive to LPS is used herein to characterize tissue immunogenicity of placental membranes at each critical manufacturing step. Samples from each of the manufacturing steps included a nutrient (T), amniotic membrane and chorionic villus (ACT), chorionic villus (CT), chorionic membrane (CM), and amniotic membrane (AM).

Placental membrane fragments (2 cm x 2 cm) representing the intermediate and final products were placed in tissue culture media and exposed to bacterial LPS (1 μg / mL) for 20-24 hours. The tissue culture supernatant was then collected and then tested for the presence of TNF- [alpha] using the TNF-alpha ELISA kit (R & D Systems) according to the manufacturer's protocol. Human hPBMC (SeraCare), known to contain monocytes reactive with LPS by secretion of high levels of TNF-a, was used as a positive control. HPBMC and placental tissue without LPS were included as controls in the analysis. In this assay, TNF-a detected in culture media in excess of about 70 pg / cm2 (corresponding to 280 pg / mL) for both spontaneous and LPS-induced TNF-a secretion was considered immunogenicity (Fortunato, et al. 1996).

As shown in FIGS. 10A and 10B, the manufacturing process continuously reduces the immunogenicity of the placental product. AM and CM had only 23.5 and 40 pg / ml TNF-a secretion at 1397.1 and 917.2 pg / ml compared to ACT and CT, respectively. Tissue cultured in LPS-free medium represents the basal level of TNF-a secretion. PBMCs known to secrete high levels of TNF were used as positive control.

The chorionic villus (CT) contains the chorion together with a deficient hypertrophic membrane. CT membranes secreting high levels of TNF-a were tested in the MLR for two different PBMC donors (Figure 11). CT cells were cultured with PBMC for 4 days. IL-2R [alpha] was measured in cell lysates as a marker of T-cell activation. Positive Control: A mixture of PBMCs derived from two different donors. As can be seen in Figure 11, the results of this analysis correlate with MLR data: tissues that produce high levels of TNF-a in response to LPS are immunogenic in MLR analysis.

In conclusion, the absence of a response to low levels of TNF-α and LPS by AM and CM indicates that the exemplary cryopreservation method described in this technique removes viable functional macrophages from the amniotic membrane and chorion, Indicating that the safety of the product is guaranteed.

Characterization of Cells in the Placental Membrane

Example 11 Analysis of placental cells by fluorescence activated cell sorting (FACS)

Knowing the cellular composition of the amniotic membrane and chorion are important for studying a thorough understanding of the potential functional roles in wound healing and immunogenicity. Previous reports have demonstrated that both amniotic membrane and chorionic membrane contain multiple cell types. In addition to stromal cells identified for both amniotic membrane and chorion, amniotic membrane also contains epithelial cells. Fetal blood vessels are absent in either the amniotic membrane or chorion, but the membranes contain both fetal macrophages in which they reside. Proximity to maternal blood circulation and decidual membranes provides a potential source of immunogenic cells (monocytes and trophoblast cells) and is thus a potential source of immunogenicity. Fluorescence activated cell sorting (FACS) analysis was performed to investigate amniotic and chorionic cell compositions. The data demonstrate the presence of stromal cells in fetal epithelial cells for amniotic membrane, and stromal cells for chorion. One unique feature of the presently disclosed placental composition is the presence of MSCs (in addition to epithelial cells and fibroblasts) which appears to be one of three cell types important for wound healing.

Example 11.1 FACS Procedure: Single cell suspension preparation

Purified amniotic membrane and chorion were used for cell phenotype analysis via FACS. Cells from the amniotic membrane and chorion were isolated using 280 U / mL collagenase type II (Washington, catalog # 4202). Tissue was treated with enzyme at 37 ° C ± 2 ° C for 60 to 90 minutes and then the resulting cell suspension was filtered through a 100 μm filter to remove tissue debris. The single cell suspension was then centrifuged at 2000 rpm for 10 minutes using Beckman, TJ-6 and washed twice with DPBS. After discarding the supernatant after each wash, the cells were resuspended in 2 mL of FACS staining buffer (DPBS + 0.09% NaN ₃ + 1% FBS).

Example 11.2 Immunoassay for specific cell markers

Once a single cell suspension was prepared according to Example 11.1, at least 1x10 &lt; ⁵ &gt; cells in 100 [mu] L of FACS staining buffer were treated with a fluorescent dye labeled antibody. Table 7 provides a description of the antibodies used. For cell surface markers, cells were incubated with antibody for 30 minutes in darkness room temperature, then centrifuged at 1300 rpm for 5 minutes using Beckman TJ-6 centrifuge and centrifuged twice using FACS staining buffer And washed. The cells were then resuspended in 400 [mu] L of FACS staining buffer and then analyzed using a BD FACScalibur flow cell analyzer. To assess cell viability, 10 μL of 7-AAD (7-amino-actinomycin D) reagent (BD, catalog number 559925) was added immediately after the initial FACS analysis and analyzed again. For intracellular staining, cells were infiltrated and labeled according to manufacturer's recommendations (BD Cytofix / Cytoperm, catalog number 554714) and analyzed using a BD FACS caliber flow cytometer.

Example 12 Expression Analysis of Placental Cells

FACS analysis of single cell suspensions of both amniotic membrane and chorionic membrane demonstrates that both membranes contain cell expression markers specific for hepatocyte stem cells involved in the presence of MSC, such as CD105 and CD166 (refer to Table 7). In addition, very few placenta macrophages expressing CD14 were detected. Some immunogenicity markers that are more likely to be expressed on CD14 + placental macrophages were detected, but these marker ranges are very broad. These ranges can be explained by: 1) high variability of cell numbers between placental donors; And 2) a technical problem involving the presence of high and variable cellular and tissue debris in the cell suspension. Debris particles similar in size to cells that can be opened or closed will affect the accuracy of the% calculated for each test marker. . Table 8 shows the characterization of the cellular composition of the placental membrane based on the selective CD marker. In addition, Table 9 provides FACS analysis of cells isolated from amniotic and chorioamnion cultures cultured in 10% FBS in DMEM at 37 ° C ± 2 ° C to confluence (passage 0 cells).

These data demonstrate that cells from amnion and chorionic villus possess a phenotype similar to MSC after incubation. In conclusion, the presence of stromal cells in placenta tissue was confirmed by FACS analysis.

These data are consistent with certain embodiments of the present technology to provide a placental product comprising amniotic membrane containing MSCs.

Optional CD On the marker Based Placental membrane  Characterization of cell compositions. Marker Amniotic membrane (range%) Chorion (range%) MSC marker CD105
CD166 72.1-88.2
17.3-58.0 6.4-78.5
4.8-51.5 Hematopoietic cell marker CD14
CD45 6.93-10.5
4.4-9.9 0.9-6.1
4.6-14.7 Immune Ball -
Stimulating factor HLA-DR
CD86
CD40 0-5.6
24.3-49.6
7.0-68.7 0-14.7
4.9-22.5
2-5.8 Nutrition marker Cytokeratin-7 1.36-4.66 2.71-23.07

placenta Lot  D16 to cultured cells ( Pass 0) of FACS  analysis. Cell surface marker amnion(%) Chorion (%) CD45 2.18 0.53 CD166 92.77 82.62 CD105 83.02 86.73 CD49a 92.28 92.26 CD73 89.57 94.57 CD41a -0.03 -0.05 CD34 -0.23 -0.25 HLA-DR -0.23 -0.19 CD19 -0.19 -0.22 CD14 -0.25 -0.27 CD90 99.12 98.00

Example 13 Adhesion of cells derived from placenta products.

In addition to the presence of specific cell markers, it can be classified as an MSC if it exhibits adhesive properties under normal culture conditions and has fibroblast-like morphology. Cells were isolated from amniotic membrane and chorion as described in this technique and plated in MSC medium and then incubated at 37 ° C ± 2 ° C until they reached confluence. Then, their ability to be adhered to a stick culture dish was evaluated.

Figure 12 demonstrates plastic adhesion of cells isolated and cultured from amniotic membrane (Figure 12a) and chorion (Figure 12b), similar to isolated and expanded MSCs from human bone marrow ash (Figure 12c). These data indicate that cells derived from the amniotic membrane and chorionic villus possess a phenotype similar to MSC.

In addition, MSCs are also represented by their ability to differentiate into different connective tissue types. Thus, the ability of placenta-derived cells to undergo osteogenic differentiation was tested. Placental cells were isolated and then incubated at 37 ° C ± 2 ° C until they reached confluence. The osteogenic medium was then added to the culture and the expression of alkaline phosphatase was measured. Alkaline phosphatase is an enzyme involved in the inorganic chloride of bone and is a well known bone formation marker. Figure 12d shows that some cells are stained with a purple dye that exhibits alkaline phosphatase expression. This demonstrates that MSCs present in placental membranes retain their potential for differentiation.

Therapeutic factor

Example 14 Cryopreservation does not impair therapeutic factor levels in placental membranes

The inventors first investigated whether cryopreservation affects the level of growth factors important for wound healing. Vascular endothelial growth factor (VEGF) was chosen because it is important in promoting angiogenesis. VEGF was extracted using incubation with 8 M guanidine hydrochloride (GuHCl) solution, bead homogenizer, and GuHCl solution for 24 hours. VEGF expression from new amnion and cryopreserved amniotic membrane products (as described by the present technology) was measured using enzyme-linked immunosorbant assay (ELISA) according to the manufacturer's protocol. The results in Figure 13 show that there is no loss of growth factor expression relative to the new membrane by cryopreserving the membrane using the procedure described in the art.

Example 15 Analysis of therapeutic factors

Protein profiles of amnion and chorion were studied using a SearchLight Multiplex chemiluminescence array or ELISA. The presence of proteins secreted by tissue membrane extracts and by tissue in culture media was studied. The test consisted of protein analysis important for wound healing. Table 10 lists the identified proteins.

Example 15.1 Preparation of amniotic membrane and chorionic villus samples for therapeutic factor profiling

Amniotic membrane and chorion were isolated and packaged at -80 ° C ± 5 ° C according to the manufacturing protocol described in Examples 1 and 2 herein. The packaged membrane was then thawed at 37 占 폚 占 2 占 폚 water bath and then washed three times with DPBS. The membrane was cut into 8 cm 2 pieces. For the tissue melt sample, one 8 cm 2 piece of membrane was instantaneously frozen in liquefied nitrogen and ground using a mortar. The pulverized tissue was transferred to a 1.5 mL microcentrifuge tube and then 500 μL of a lysis buffer (cell signaling technique, Catalog No. 9803) was added with a protease inhibitor (Roche, catalog number 11836153001) and then incubated for 30 minutes Lt; / RTI &gt; on ice. The tissue lysate was then centrifuged at 16000 g for 10 minutes. The supernatant was collected and then sent for protein array analysis by Aushon Biosystems. For the supernatant sample, one 8 cm 2 piece of membrane was plated into the wells of a 12-well dish and then 2 mL of DMEM + 1% HSA + antibiotic / antifungal agent was added followed by 3, 7 or 14 Lt; / RTI &gt; After incubation, the tissue and culture medium was transferred to a 15 mL centrifuge tube and then centrifuged at 2000 rpm for 5 minutes. The culture supernatant was collected and then sent for protein array analysis by Oshon Biosystems.

Example 15.2 Therapeutic Factors Present in Tissue Seawater

Placenta product lysates were analyzed for the presence of proteins important for tissue repair. Table 11 shows the biochemical profile of a lysate of an exemplary placental tissue product of the art.

Example 15.3 Continuous release of therapeutic agent over a period of 14 days

The placental products of the present technology also demonstrate a sustained effect desirable for wound healing therapies. The presence of extracellular matrix and viable cells in the amniotic membrane described herein allows cocktails of proteins known to be important for wound healing to be provided for at least 14 days. The amniotic membrane was thawed, plated on tissue culture wells, and incubated at 37 ° C ± 2 ° C for 3, 7 and 14 days. At each time point, culture supernatant samples were collected and then measured by protein array analysis as described in Example 15.1. Table 12 illustrates various selective factor levels in tissue culture supernatants of amniotic membrane lots at 3, 7 and 14 days as determined by protein array analysis.

Example  15.4 Amniotic interferon 2α ( IFN - 2α ) And transforming growth factor- β3  (TGF-β3)

Interferon-a and TGF-b3 are cytokine / growth factors that are known to reduce fibrosis in a variety of tissues. Clinically, IFN-2α and TGF-β3 have been implicated in the regulation of wound healing by preventing scarring and build-up (Ishida, Kondo et al., 2004; Ferguson, Duncan et al. IFN-2alpha inhibits fibroblast proliferation and may play a role in reducing collagen and fibronectin synthesis and fibroblast-mediated wound formation (Wang, Crowston et al. 2007). Clinically, IFN-2 alpha has been shown to be administered subcutaneously and to improve scarring (Nedelec et al, Lab Clin Med 1995, 126: 474). TGF-β3 modulates the deposition of extracellular matrix and reduces scar formation when injected in a rodent transcutaneous wound model. Clinically, TGF-β3 has been shown to improve scarring when injected at wound sites (Occleston et al., J Biomater Sci Polym Ed 2008, 19: 1047). TGF-β3 functions as a TGF-β1 antagonist that regulates fibroblast-myofibroblast differentiation and limits profibrotic gene transcription (Chang, Kishimoto et al. 2014).

The placental products described in this technique were analyzed for the presence of IFN-2 alpha and TGF- beta 3. Briefly, after thawing, the membranes are homogenized and the supernatant obtained by centrifugation at 16,000 g is collected. The supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2 alpha) and from Ald Systems (TGF- beta 3). Figure 14 shows significant expression of IFN-2? (A) and TGF-? 3 (B) in the placental product cell lysate.

Example  15.5 Amniotic membrane and chorionic membrane My bFGF  existence

bFGF regulates a variety of cellular processes including neovascularization, tissue repair and wound healing (Presta et al., 2005, Reuss et al., 2003, and Su et al., 2008). In wound healing models, bFGF has been shown to increase wound closure and improve angiogenesis at wound sites (Greenhalgh et al., 1990). Evaluation of proteins derived from the amniotic membrane and chorion from the cholestatic membranes prepared by the presently disclosed production method indicates that bFGF is one of the major factors in placental tissue protein extract. Figure 14B shows the expression of bFGF by amniotic membrane (AM) and chorionic membrane (CM) from two donors detected during protein profile evaluation of the placental membrane.

Example  15.6 Amniotic placental growth factor PLGF ( PLGF ) And the presence of insulin-like growth factor-1 (IGF-1)

Without being bound by theory, the inventors believe that the efficacy of the present placental product for wound restoration is due in part to the role of BMP, IGF-1 and PlGF in the development and homeostasis of various tissues by modulating cellular processes that are important Believe. BMP-2 and BMP-4 can stimulate the differentiation of MSC into osteoblasts in addition to promoting cell growth; Placental BMP or PLAB is a novel member of the BMP family suggested to mediate embryonic development. Insulin-like growth factor-1 (IGF-1) can promote the proliferation and differentiation of bone progenitor cells. Placenta-derived growth factor (PlGF) can act as a mitogen for osteoblasts.

The placental products described in this technique were analyzed for the presence of tissue repair proteins. Briefly, the thawed product was incubated in DMEM + 10% FBS for 72 hours. The membranes were then homogenized in a bead homogenizer with the culture medium. The cell lysate was centrifuged and then the supernatant was analyzed on a commercially available ELISA kit from Alded Systems.

Figure 15 shows significant expression of BMP-2, BMP-4, BMP-7, PLAB, PlGF and IGF-1 in some donors of the amniotic membrane.

Example 15.7 Presence of? 2-macroglobulin in the amniotic membrane

α2-macroglobulin is a plasma protein that deactivates proteases (serine protease, cysteine protease, aspartate protease and metalloproteinase) from all four mechanistic classes It is known. Other important functions of this protein function as a reservoir of cytokines and growth factors, examples of which include TGF, PDGF and FGF. In chronic wounds such as diabetic ulcers or vein ulcers, the presence of high amounts of protease causes rapid degradation of growth factors and delayed wound healing. Thus, the presence of [alpha] 2-macroglobulin in the product designed for chronic wound healing would be advantageous. Protein array analysis results indicate that amniotic membrane and chorionic membrane contain alpha 2-macroglobulin (Table 13). Although these preliminary data show high variability among donors, the importance of this protein in wound healing has prompted an additional assessment of a2-macroglobulin in placental tissue using a single analyte ELISA instead of a protein array, It is a useful tool for assessing the presence of multiple proteins in one sample.

These data are consistent with certain embodiments of the present technology to provide placental products comprising amniotic membrane containing alpha 2-macroglobulin.

Expression of α2-Macroglobulin in Placenta Tissue Protein Extract. Sample α 2-macroglobulin (pg / mL / 8 cm 2) AM75 7 CM75 790 AM78 53042 CM78 1014

Example 16 Comparison of Therapeutic Parameters in Illustrative Placental Tissue and Two Commercially Available Products

For comparison, profiles of two commercially available products containing living cells, Dermagraft and Applescript were similarly analyzed using a SearchLight Multiplex chemiluminescent array.

Example 16.1 Protocol for comparison of exemplary placental tissue and two commercially available products

To test the dermagraft, the membranes were thawed and washed according to the manufacturer's instructions. The Durma graft membrane was cut into 7.5 cm 2 pieces. For the tissue lysate, one 7.5 cm 2 piece of membrane was instantaneously frozen in liquefied nitrogen and ground using a mortar bowl. The pulverized tissue was transferred to a 1.5 mL microcentrifuge tube and then 500 μL of a lysis buffer (cell signaling technique, Catalog No. 9803) was added with a protease inhibitor (Roche, catalog number 11836153001) and then incubated for 30 minutes Lt; / RTI &gt; on ice. The sample was then centrifuged at 16000 g for 10 minutes. The supernatant was collected and then sent for protein array analysis by Aushon Biosystems. For the tissue culture, one 7.5 cm 2 piece of membrane was plated into the wells of a 12-well plate followed by the addition of 2 mL of DMEM + 1% HSA + antibiotic / antifungal agent followed by 3, 7, Lt; / RTI &gt; for 14 days. After incubation, the tissue and culture medium was transferred to a 15 mL centrifuge tube and then centrifuged at 2000 rpm for 5 minutes. The culture supernatant was collected and then sent for protein array analysis by Oshon Biosystems.

To test the application, the membrane was cut into 7.3 cm 2 pieces. For tissue lysates, one 7.3 cm2 piece of membrane was instantaneously frozen in liquefied nitrogen and ground using a mortar bowl. The pulverized tissue was transferred to a 1.5 mL microcentrifuge tube and then 500 μL of a lysis buffer (cell signaling technique, Catalog No. 9803) was added with a protease inhibitor (Roche, catalog number 11836153001) and then incubated for 30 minutes Lt; / RTI &gt; on ice. The sample was then centrifuged at 16000 g for 10 minutes. The supernatant was collected and then sent for protein array analysis by Aushon Biosystems. For the tissue culture, one 7.3 cm 2 piece of membrane was plated into the wells of a 12-well dish and then 2 mL of DMEM + 1% HSA + antibiotic / antifungal agent was added followed by 3, 7, Lt; / RTI &gt; for 14 days. After incubation, the tissue and culture medium was transferred to a 15 mL centrifuge tube and then centrifuged at 2000 rpm for 5 minutes. The culture supernatant was collected and then sent for protein array analysis by Oshon Biosystems.

Example 16.2 Third Day of Example Placental Tissue and Commercially Available Product Therapeutic agents present in the supernatant

Protein array data analysis expressed the majority of the selected test agents (designated Table 11) in amniotic membrane, chorionic villus, Applescript and Derma graft. Three proteins were identified as unique to the amniotic membrane and / or chorionic membrane which were not detectable in the appliGraphs and Derma grafts. These proteins are EGF, IGFBP1 and adiponectin. All three proteins are important for wound healing. Figure 16 shows the expression of EGF (a), IGFBP1 (b), and adiponectin (c) in amniotic membrane (AM), chorionic membrane (CM) and commercially available products. AM75 and AM78 are cryopreserved placenta products of the present technology (e.g., cryopreserved) whereas CM75 and CM78 are cryopreserved chorionic villus products. These proteins are believed by the inventors to enable therapeutic efficacy of the present placenta product for wound healing.

These data are consistent with certain embodiments of the present technology to provide a placental product comprising amniotic membrane containing EGF, IGFBP1, and / or adiponectin.

Table 14 shows the biochemical profile of the exemplary placental product supernatant and the two commercially available products of the present technology (the result being adjusted every 8 cm 2 after subtraction of the negative background). AM75 and AM 78 are placental products of the technology (e.g., cryopreserved), and CM75 and CM78 are cryopreserved chorionic villus products.

Both MMP and TIMP are important factors in wound healing. However, the expression of these proteins must be highly regulated and regulated. Excessive MMP for TIMP is a marker of poor chronic wound healing. We studied the expression of MMP and TIMP and their ratio in amniotic membrane and chorion, and compared them to expression profiles in Applescript and Dermagraft.

The results in Table 14 and Figure 17 show that all membranes express MMP and TIMP; However, it was shown that the ratio in the thawed placenta product and chorion was significantly lower. Thus, these membranes would be more beneficial for wound healing (Figure 17).

The accumulated data indicate that the MMP versus TIMP ratio is higher in the case of non-healing wounds. For example, the ratio between MMP-9 and TIMP1 is approximately 7 to 10 for good healing and 18 to 20 or more for poor healing. Analysis of the ratio between MMP and TIMP secreted by placental tissue, Applescript and Derma graft indicates that amniotic membrane and chorionic villus products contain MMP and TIMP at approximately the ratio 7 desirable for wound healing. In contrast, the Dermagraft has a ratio of more than 20, and Apples has a ratio of more than 200.

These data are consistent with certain embodiments of the present technology to provide a placental product comprising amniotic membrane containing MMP-9 and TIMP1 at a ratio of about 7 to 10: 1.

Example 17 Establishment of EGF as a marker for amniotic membrane efficacy

EGF is an important factor in wound healing (Schultz et al., 1991, Komarcevic, 2000, and Hong et al., 2006). The absence or reduced amount of EGF is one characteristic of chronic wounds (Harding et al., 2002). The evaluation of amniotic membrane-derived proteins produced according to the developed manufacturing process disclosed by the present application indicates that EGF is one of the major factors secreted by these tissues in a larger amount. The importance of EGF for wound healing with the high level of EGF detected in the amenoriminally disclosed amniotic membrane supports the selection of EGF as an efficacy marker for the evaluation of membrane products prepared for clinical use by this disclosure. Commercially available ELISA kits from R & D Systems Inc. were selected for evaluation of their suitability for measuring amniotic membrane secreted EGF. ELISA quantification meets the standards established by the FDA and the ICH Guidelines on the validity of biological assay assays (ICH Harmonized Tripartite Guideline and Guidance for Industry Bioanalytical Method Validation, 2001 ). Amniotic membranes evaluated for expression of EFG by this method identified protein array data and further demonstrated that EGF was a unique factor expressed at clinically significant levels in these tissues.

Example 17.1 Amniotic Membrane Tissue Expression of EGF

Protein array analysis provided early evidence that EGF was uniquely expressed in the amniotic membrane, but not in the chorion (Table 15). The level of EGF measured in the amniotic membrane has clinical significance.

Protein array data showing the expression range of EGF in amniotic membrane and chorionic membrane from multiple donors Amniotic membrane (pg / ml) Chorioamnion (pg / ml) EGF 127.3-361.4 0-0.8

These data are in accordance with certain embodiments of the present technology to provide a placental product comprising amniotic membrane optionally containing EGF in substantial amounts.

Cell lysate of placenta product

The placenta product was thawed until no ice crystals were present. The membrane was then removed from the bag and then cut into 4 cm x 2 cm pieces while still adhered to the nitrocellulose. Each piece of tissue was then removed from the nitrocellulose and then washed twice with PBS. Each tissue was then instantaneously frozen in a homogenization tube with liquid nitrogen. Subsequently, one pre-frozen 5 mm steel bead was added to each tube and then the sample was homogenized in a 500 [mu] L homogenization medium using a Qiagen Tissue Lyser according to the manufacturer's recommendations . Tissue cell lysates were stored at -80 ° C ± 5 ° C until analyzed by ELISA for EGF expression.

Amniotic membrane EGF  Expression

Measurement of EGF in amniotic membrane preparation has proven to be reliable and reproducible. Measurement of EGF in a number of donors has shown that this quantification method is a valuable method for assessing the efficacy in tissues produced by this disclosure for use in clinical situations. Figure 18 shows representative expression of EGF in thawed placenta products and chorions produced and analyzed by the methods described above. The results were reproduced in multiple tissue preparations.

These data are consistent with certain embodiments of the present technology to provide a placental product comprising amniotic membrane containing EGF.

Functional studies:

Example 18 Immunoregulatory Effect of Placental Membrane

Chronic wounds do not progress through the normal healing phase and are often stopped at the inflammatory stage (Maxson, Lopez et al. 2012). The characteristics of the chronic wound environment are as follows: 1) a high level of proinflammatory cytokines such as TNF-α and IL-1α, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, High levels of oxidizing agents and low levels of antioxidants. Thus, inflammation control at the site of injury is the key to resuming / proceeding the healing process. The efficacy of placental membranes for antiinflammatory activity was studied for wound healing.

Example 18.1 Tumor necrosis factor alpha (TNF-a) stimulation

Tissue samples were incubated for 18 hours with or without 1 ng / ml TNF-alpha (proinflammatory factor). After collecting the supernatant, the concentration of prostaglandin E2 (PGE2) was measured using a PGE2 monoclonal enzyme immunoassay (EIA) kit (Cayman). Figure 19 shows that our placental membrane product (amniotic membrane product) produces a high level of anti-inflammatory cytokine PGE2 when exposed to TNF- [alpha].

Example 18.2 Peripheral Blood Mononuclear Cell (PBMC) Analysis

PBMC were obtained from SeraCare Life sciences. All experiments were performed in duplicate in 24 well plates with 10 ⁶ mononuclear cells in 1 mL assay medium per well. Immune cells were activated by adding T cells mitogen-anti-CD3 monoclonal antibody (CD3) and anti-CD28 monoclonal antibody (CD28) at 1 μg per mL to test the inhibitory effect of proinflammatory cytokines. Subsequently, the tissue samples were incubated with a humid atmosphere 48 hours 37 ℃ and activated PBMC in 5% CO ₂ for from. TNF- [alpha] and IL-l [alpha] production was measured in supernatants using an ELISA Duoset kit (R & D Systems). To test the release of the anti-inflammatory factor IL-10, we pre-stimulated PBMC with 100 ng / ml LPS for 4 hours and then incubated tissue samples with stimulated PBMC for an additional 24 hours (10 ng / mL Pre-stimulation overnight with TNF-a). IL-10 was detected in the supernatant using an IL-10 Duo Set Kit (R &amp; D Systems). PBMCs without LPS stimulation were included as negative controls. The placental membrane products significantly inhibited the release of pre-soluble inflammatory cytokines (TNF-a, and IL-l [alpha]) and upregulated the release of anti-inflammatory IL-10 when co-cultured with activated immune cells Lt; / RTI &gt;

Example 18.3 Modulation of elevated levels of protease by amniotic membrane

The prolonged inflammatory response in chronic wounds results in an enhanced protease response, especially with increased MMP and neutrophil elastase activity. MMP and elastase are implicated in normal physiological and pathological processes such as degradation of basement membrane, remodeling of ECM, connective tissue conversion, neovascularization, regeneration and wound restoration. However, excess MMP and elastase destroy the ECM components and damage growth factors essential to healing and their receptors. In this study, we studied whether placental membrane products mediate inhibition of MMP and elastase.

Azocol analysis for MMP activity

Azocol is an insoluble, crushed collagen impregnated with a purple azo dye. Upon proteolysis, the soluble azo dye is released and can be detected by absorbance at 550 nm. Thus, azocol is often used as a color-forming nonspecific substrate for testing protease activity in the environment. The analysis was performed using a transformation method developed by Jiang et al. (Jiang, Tan et al. 2007). The azocol is washed and then suspended in 10 mM PBS, pH 7.4, to a final concentration of 1.5 mg / mL. Collagenase IV was used as a positive control, indicating that its two active forms, MMP-2 and MMP-9 (72 kD and 92 kD, respectively), were elevated in wound from chronic leg ulcers associated with poor healing (Trengove, Stacey et al. 1999). Amniotic membrane products were incubated with 0.1% (w / v) collagenase IV (Life Technologies) and azocol suspension for 5 hours under rotation from a soft end to a tip in a 37 ° C incubator. The reaction was stopped by centrifuging the sample at 10,000 xg for 8 hours. The absorbance of the supernatant solution was measured at 550 nm using an ELISA reader (spectramax) and a statistical significance was determined by performing a two-sided Student t test (p < 0.05). Figure 21 demonstrates that cryopreserved amniotic membrane can significantly inhibit MMP activity.

Neutrophil elastase analysis

The amniotic membrane product was preconditioned with 100 ng purified human neutrophil elastase (Sigma) in DMEM for 24 hours with 1% FBS. Pre-conditioned tissue samples and 100 ng of fresh neutrophil elastase were incubated for 4 hours at 37 DEG C in a final volume of 500 l of 0.1 M HEPES buffer containing 0.5 M NaCl, 10% DMSO and 1 mM elastase substrate (Sigma), pH 7.4 &Lt; / RTI &gt; Substrate degradation was monitored continuously by measuring OD ₄₀₅ . Figure 22 shows that the cryopreserved amniotic membrane inhibited elastase activity by approximately 94%.

Example  18.4 In the wound ROS  Neutralization

Immune cells such as neutrophils and macrophages produce reactive oxygen species (ROS). Low concentrations of ROS provide signaling and defense against microorganisms. However, large amounts of ROS not only damage extracellular structural proteins, lipids and DNA, but also enhance the expression of MMPs, serine proteases and inflammatory cytokines, which impairs wound healing. The inventors first studied the total antioxidant capacity of placental membrane products using an antioxidant ability kit. In addition, it was tested whether placental membrane products could protect dermal fibroblasts from oxidant-induced apoptosis.

Antioxidant analysis

The antioxidant activity of conditioned media from amniotic membrane products was determined using an antioxidant assay kit (Sigma CS0790) according to the manufacturer &apos; s instructions. The method is based on the release of the radical cation ABTS ⁺ from ABTS which produces a green signal when exposed to oxidative conditions. Inhibition of the resulting color intensity may be related to the antioxidant capacity of the sample. Figure 23 demonstrates that the amniotic membrane product has the stronger antioxidant ability as 250 [mu] M ascorbic acid.

Cell survival analysis

Cell viability assays were performed using the modified method reported by Kim (Kim, Park et al. 2008). To normal human dermal fibroblast (NHDF) (Lonza) in a 24-well plate for 5 x 10 ⁴ cells / well plated at a density of, and 24 hours in a DMEM supplemented with 0.1% FBS nutrition was blocked. Subsequently, tert-butyl hydroperoxide (tbOOH), an organic hydroperoxide, was used as an oxidizing agent to induce oxidative damage to NHDF for 3 hours. After replacing the tbOOH containing medium with the normal culture medium, the amniotic membrane product is incubated with HDF to start the rescue procedure. The positive control was replaced with 250 [mu] M ascorbic acid. After 4 hours, NHDF was incubated with 10 [mu] g / ml Hoechst for 30 min at 37 [deg.] C, and by fluorescence microscopy using ImageJ (NIH) for cells with strongly condensed chromatin and / or fragmented nuclei Apoptosis was determined by scoring percentages. Figure 24 demonstrates that amniotic membrane products can up to 80% of their initial apoptotic HDF.

Example  19 Placenta membrane  The product was used to induce angiogenesis Improve

Neovascularization is a critical step in the wound healing process. The formation of new blood vessels is essential to provide fibroblasts with sufficient nutrient supply for the production of transient granulation substrates and the survival of keratinocytes.

Tube formation

To test the potential for angiogenesis of amniotic membrane products, HUVECs were immunized with amniotic membrane-derived conditioned media derived from amniotic membrane products at a concentration of 10 ⁴ cells / well on Matrigel (BD) -coated culture wells Lt; / RTI &gt; Conditioned media were obtained by culturing amniotic membrane products in endothelial growth medium EBM (longe) supplemented with 2% FBS for 3 days. EBM supplemented with all essential cocktails of growth factors was used as a positive control. After 8 hours of incubation, the fields from each sample were randomly photographed by an inverted microscope, the number of sealants was digitized and then plotted by Image J (NIH).

Figure 25 demonstrates that the conditioned medium from the amniotic membrane product improves tube formation as with the positive control.

Example  20 Placental tissue can be used for cell migration and wound healing Improve

The wound healing process is highly complex and involves a series of structured events controlled by growth factors (Goldman, 2004). These events include increased angiogenesis, infiltration by inflammatory immune cells, and increased cell proliferation. The initiation phase of wound healing deals with the ability of individual cells to become polarized to the wound, to move into the wound area to approach the wound area and reconstitute the surrounding tissue. Three types of cells are involved in the process of cell migration. They are vascular endothelial cells, fibroblasts and keratinocytes. Vascular endothelial cells migrate to the wound area and form new blood vessels that provide essential oxygen and nutrients for proper wound healing. Fibroblasts need to migrate to the wound site to form granular tissue to reconstitute various connective tissue components. Re-epithelialization (wound closure) requires directed migration of keratinocytes. Thus, the inventors sought to determine whether factors secreted from the amniotic membrane and chorion, produced in Osiris, promote three types of cell migration and wound field suture. To achieve this, we used commercially available wound healing assays as can be seen in Figure 26 (Cell Biolabs) and transwell cell migration assays. The cell lines are derived from human microvascular endothelial cells (HMVEC, Lonza Inc.), human umbilical endothelial cells (HUVEC, Lonza Inc.), human dermal fibroblasts (HDF, Lonza Inc.) &Lt; / RTI &gt; diseased keratinocytes (D-HEK Theoreticians). The results indicate that cell migration of all three types of cells is enhanced by treatment with conditioned media from placental membranes.

Example 20.1 Placental Membrane Conditioning Medium Supports Endothelial Cell Migration and Wound Field Suture

Cells were harvested through trypsinization, pelleted, digested, and resuspended in complete keratinocyte at a density of ² x 10 ⁵ cells / mL. The cell suspension of 250 ul (5 x 10 ⁴ cells) is then pipetted onto each side of the well containing wound healing insert (cytoselect 24-well wound healing assay plate, Cell Biolabs). Cells were grown in complete medium for 24 hours. After 24 hours, the wound insert is removed. At the same time, the complete keratinocyte medium is removed and replaced with the experimental medium. Complete keratinocyte and basal keratinocyte media were used as positive and negative controls, respectively. To make the experimental medium, placenta membranes are incubated in DMEM for 3 days with 1% human serum albumin (HSA) in tissue culture incubator. Subsequently, the obtained tissue and culture medium were placed in an Eppendorf tube and then rotated at a high speed in a microcentrifuge. The supernatant was collected and stored at -80 ° C until use. For migration and wound healing studies, the conditioned media from the placenta membranes is diluted 1:20 in basal keratinocytes and then added to the experimental wells. After 18 hours, the medium is removed and the cells are fixed in 4% paraformaldehyde for 20 minutes and stained with crystal violet. The wound field is then photographed in each well. Wound healing as determined by the amount of wound field is still visible at the end of the experiment as compared to control photography taken before the conditioned medium is added to the wells.

Control media from amniotic membrane and chorion were used to assess the ability of these membranes to promote endothelial cell migration and wound field suture. The conditioned medium from the combination of placenta amniotic membrane, chorion, and amniotic membrane / chorioamnium supported endothelial cell migration into the experimental wound field (Figure 27).

Example 20.2 Placental membrane conditioning media supports endothelial cells, fibroblasts, and keratinocyte migration.

Transwell cell migration assay

Mobility analysis was performed on human umbilical vein endothelial cells, human normal dermal fibroblasts, and human disease keratinocytes (type II diabetes) (loner) using a fluoro block transwell system using 8 mu m pore (BD). 100,000 cells were suspended in DMEM with 0.1% FBS and then added to the upper chamber of the transwell to inhibit mitosis. The following day, conditioned media derived from tissue samples was added to the lower chamber and then incubated overnight. After incubation, the cells migrated through the filter were fixed and stained against Carlsein (Molecular Probe). The field was visualized by a fluorescence inversion microscope and then photographed from four randomly selected fields under 10x magnification. The results of the present inventors have demonstrated that the amniotic membrane product promotes the migration of endothelial cells (FIGS. 27 and 28), fibroblasts (FIG. 29), and keratinocytes (FIG. 30).

Clinical Research

Example 21 Uses of Placenta Products to Treat Diabetic Foot Ulcers

OBJECTIVES: Despite the biologically treated skin substitutes containing human fibroblasts or a combination of human fibroblasts and keratinocytes, the published rate of chronic wound healing has been reduced to almost half of all wounds resistant to these new therapies It remains low. The morbidity and mortality from diabetic foot ulcers is substantial as the 5-year mortality rate is 39% to 68% after limb amputation. (Page J. J Foot & Ankle Surgery 2002; 41 (4): 251-259; Isumi Y., et al. Diabetes Res and Clin Practice 2009; 83: 126-131).

A cryopreserved placental membrane product providing essential angiogenic and antiinflammatory growth factors was introduced in an effort to improve the outcome of patients with chronic skin ulcers at risk of cleavage.

PURPOSE : Patients with chronic diabetic foot ulcers that are unreactive to available therapies and at risk of cleavage have been considered for treatment. All wounds were aggressively removed prior to application of frozen placental membranes. The patient is assessed periodically and the application of the membrane product of the present technology is at the discretion of the treating physician. Offloading was recommended in both patients.

Introduction

According to the United States Food and Drug Administration (FDA), chronic, skin ulcers are defined as wounds that have not progressed through a series of timely and timely events that produce sustainable structural, functional, and cosmetic sutures. (2). The most common chronic wounds include bedsores and leg ulcers. Triad of peripheral neuropathy, anomalies and minor trauma were the most frequent causes of seizures leading to foot ulcers. For healing rates, an appropriate benchmark for chronic wounds is a size reduction of 10-15% per week, or a 50% reduction in size over a one month period. A 3-year posterior cohort study performed by Ramsey et al in 8,905 patients with diabetes mellitus showed a 5.8% cumulative incidence of ulcer. At diagnosis, 15% of these patients developed osteomyelitis, and 16% required partial cleavage of the lower limb.

Approximately 80% to 85% of limb amputations are progressed by foot ulcers. The morbidity and mortality from diabetic foot ulcers are substantial as the 5-year mortality rate after limb removal is 39% to 68%. (2). These mortality rates are higher than the 5-year mortality rate for breast cancer, colon cancer and prostate cancer.

Despite all advances in biotechnology-treated tissues for the treatment of chronic diabetic ulcers, many patients are resistant to treatment and have ulcers that cause chronic scarring. Because of the healing rate approaching only 50% of these newer therapies, the use of stem cells in regenerative medicine has recently become of particular interest. The ultimate goal is to promote the recovery of functional skin. Preliminary studies were performed by Fiami et al., Which isolated hepatocellular stem cells from umbilical cord blood and inoculated them into de-epithelialized dermis pieces. This preliminary study indicates that peripheral stem cells can survive and express neovascularization. In addition to indicating the potential for tissue repair, hepatic stem cells exhibit low immunogenicity and can be universally transplanted without undergoing a compatibility test between donor and recipient.

In this study, clinical evidence of significant healing utilizes cryopreserved membrane products for the treatment of two chronic wounds in which cleavage is considered. Fundamental wound management is still the cornerstone of comprehensive wound healing in any treatment protocol including appropriate necrosis removal, offloading, maintenance of the water environment and proper perfusion and infection control.

matter

The cryopreserved placental membranes comprising native epithelial cells and allografts derived from the amniotic membrane comprising a bilayer of a substrate layer consisting of neonatal fibroblasts, extracellular matrix (ECM) and hepatic stem cell (MSC) Respectively.

Limb surgery: Case 1

History and physical examination

A 70-year-old male with vesicle formation, edema and pain on the lateral aspect of the fourth and fifth toes on the right foot and a small lesion on the side of the fifth toe came to the emergency room. The patient reported a history of minor trauma to the area two weeks before the visit. The patient had chronic kidney disease receiving type II diabetes mellitus, hypertension, heart failure, chronic obstructive pulmonary disease, and hemodialysis three times a week. The patient had a history of replacement of the aortic-vein graft. He denied any history of alcohol, tobacco or drug use. Physical examination did not show active purulent drainage or odor, and showed no smell upon stimulation. The angiogram showed pulses that could not be accelerated in the parietal and posterior tibial arteries. Doppler examinations showed monoclinic papillary arterial pulses with posterior fossa oval artery. The fifth toe had gangrene changes and was cold when accelerated. There was an ischemic change in the fourth toe. The radiographic evaluation showed sporadic air density indicating the fifth toe tip as well as the soft tissue gas of the fourth gap.

Preoperative management

The patient initiated intravenous antibiotics of vancomycin and piperacillin and tazobactam in appropriate kidney dosing.

Operation management

The patient went to the operating room where the incision and discharge of the fourth interstice was performed, and a partial fifth incision of the metatarsal level was performed without complications. The wound was left open and filled with sterile gauze moistened with sterile normal saline, then covered with sterile pressure dressing. The intraoperative findings showed fusion necrosis of peripheral tissues with pyrogen and odor. The patient underwent two subsequent follow-up surgical necrosis removal and the second caused additional removal of the fourth and fifth metatarsals. In the third operation, additional necrotic tissue removal of the necrotic soft tissue and cleavage of the fourth toe were performed. On May 20, 2010, treatment with cryopreserved placental membranes was initiated. Prior to implant placement, the patient underwent successful resumption of the dorsal and peroneal arteries without significant residual stenosis.

Postoperative course

The patient was discharged from the hospital and within two days he was followed by his foot doctor. At initial testing, there were no clinical signs of infection and proximal dorsal incision was seen to be spliced. The third toe was dark and the appearance was cold. Radiographs were taken showing no evidence of soft tissue gas or acute osteomyelitis. Dry sterile dressing was applied. Patients were covered with cryopreserved placenta membranes at six additional visits at the outpatient clinic. Before each application, the wound was assessed for abscess, ringworm, drainage, hematogenesis and infection. At each visit, the wound was reduced in size and in fact showed more granules than previous visits. In the third application, the wound was reduced in size by 50%.

At 19 weeks, the wound was sutured and the patient was instructed to maintain weight bearing on the affected limb while using only the orthodontic shoes.

A photograph of the notable wound healing is mediated by the placenta product of the technique as shown in Fig. Panel a: First application of cryopreserved placental product; b: after 8 weeks of the first cryopreserved placental membrane, apply this film product; c: after 10 1/2 weeks of the first cryopreserved placenta membrane, apply the present membrane product; d: after 12 weeks of the first cryopreserved placental membrane, apply this film product; e: After 19 weeks of the first cryopreserved placental membrane, the present membrane product is applied.

Limb salvage : Case 2

History and physical examination

A few weeks before the visit, a 44-year-old man with a large ulcer on the foot of the second left toe of the left to the previous trauma visited the outpatient clinic. The patient had a history of diabetes mellitus for the past 5 years, combined with peripheral neuropathy, hypertension, dyslipidemia, and osteomyelitis. Past history of surgery included abdominal aortic aneurysm repair and phantom resection. On physical examination, the ulcer was measured as 4.0 cm x 2.0 cm x 1.5 cm, and the tendon bone was examined. There was no synovial or lymphatic salt, and the temperature gradient was not increased. Capillary filling time, hair growth and tissue tension were all normal. There was a distinct pulse in the pedicular artery and posterior tibial artery. Radiographs were negative for soft tissue gases. Magnetic resonance imaging showed osteomyelitis in the distal aspect of the first metatarsal and tarsal bones with small soft tissue abscesses in the soft tissue area adjacent to the tarsal bones.

Preoperative management

The patient started intravenous antibiotics. He went to the operating room to remove ablative necrosis of all non-viable tissues and to apply the membrane product of the present invention.

Operation management

Resection with healthy tissue was performed by using both ulcer-sharp incisions and VERSAJET (trade name) to leave the head of the first bony bone exposed to the soles of the feet. The cryopreserved placental film product was then placed on the wound layer and the bones were exposed. The patient survived the procedure without complications. The patient was discharged from the hospital the day after surgery for the 5-week course of intravenous antibiotic therapy.

Postoperative course

The patient was instructed not to have a strictly weight bearing on the affected limb and returned for follow-up on postoperative day 6. The dressing was clean, dry and complete. There were no postoperative complications such as abscess, ringworm, discomfort, or drainage, and there were no clinical signs of infection. The patient received a total of 7 cryopreserved placental membrane products over the next 8 weeks. At each visit, we looked at clinical signs of wound infection. Each visit evaluation showed a significant reduction in the size and size of the granulation tissue on the wound substrate. After 8 weeks of initial application of allograft tissue, the wound was sutured.

A photograph of the notable wound healing is mediated by the placenta product of the technique as shown in Fig. Panel A: Investigation of osteomyelitis, exposed tendons and bones. The first cryopreserved placental graft was used after surgical necrosis; B: After a single application of the frozen placental membrane graft, the wound is virtually granular and there is no indication of infection; C: After three weeks of surgical intervention, with two applications of cryopreserved placental graft, the wound becomes significantly smaller in circumference and depth; D: After 6 weeks of surgical intervention, the wound is almost closed; At 8 weeks and 7 times of cryopreserved placental graft, the wound is sutured.

conclusion

Despite tremendous advances in skin tissue engineering in the past few decades, current therapies have limited efficacy in the treatment of chronic diabetic ulcers. As shown in this case report of both patients, the use of advanced therapies containing stem cells ultimately heals these patients instead of incising them, reduces mortality, and at the same time is a cost effective alternative to currently available standard therapies It can prove useful. Both of the patients highlighted in this case report were subjected to seven applications of the membrane product of the technology. Both patients were completely healed. Therapeutic complications were not reported and the membrane product of the present invention was safe and effective in the initial evaluation of two patients with diabetic foot ulcers at risk of incision. These results indicate that patients with resistant, chronic wounds should consider this new treatment.

Example  Evaluation of the efficacy and safety of placenta membranes for the treatment of chronic diabetic foot ulcers in future clinical trial studies

Diabetic foot ulcers result in significant morbidity and increased health care costs. This study evaluated the efficacy and safety of cryopreserved placental membranes (N = 50) compared to standard wound management (N = 47) for the treatment of chronic diabetic foot ulcers. This example illustrates the use of a cryopreserved placental membrane product (see, for example, Grafix &lt; (R) &gt;, Osirisera &lt; (R) &gt;, Columbia, Maryland) derived from amniotic membrane for the treatment of chronic diabetic foot ulcers It is part of a prospective, multicenter, randomized, single-blinded clinical trial to evaluate the safety and efficacy of paclitaxel (Osiris Therapeutics). This study followed the Ethics Guidelines of the Helsinki Declaration of 1975 and enrolled in Clinical Trials.gov (NCT01596920). The patient reviewed and signed the standard IRB consent form before enrollment. Patients were enrolled from May 2012 to April 2013.

Critical inclusion criteria include confirmed wound type I or type II diabetes, age 18 to 80 years of patient age, wound index present at 4 to 52 weeks, wound located below the radiating bone on the soles or soles of the foot and 1 cm 2 to 15 cm 2 Of ulcers. The primary exclusion criteria included evidence of an active infection, including an excess of 12% of hemoglobin A1c, osteomyelitis or ringworm, an ankle brachial index <0.70 or> 1.30, or a toe brachial index of less than 0.50, or improper circulation to the affected foot, Doppler studies by exposed muscles, tendons, bones or capsules, and a reduction of more than 30% of the wound area during the screening period.

(1) After the weekly screening period, the patients were randomized to a 1: 1 ratio for cryopreserved placenta products or control arm. Patients randomized to treatment with placenta products received placental product once (weekly) for one week (± 3 days) until day 84 (blinded treatment). Patients in the control group received one standard wound treatment for one week (± 3 days) for up to 84 days. All wounds were adequately cleaned and removed by surgery to remove all non-viable soft tissue from the wound by the scalp, tissue nipper and / or curette at each weekly visit. For patients randomized to placental product groups, the cryopreserved placental membrane was placed in complete contact with the wound and the rim. In both groups the wound received standard wound care including surgical necrosis removal, offloading and non-adhesive dressing. All patients received non-adhesive dressing: ADAPTIC (R) (Systagenix, Gatwick, UK) and saline-gauze or ALLEVYN (R) for drainage wounds of the middle ear (Smith &amp; Nephew, London, UK). Subsequently, external dressing was applied. Patients were provided with postoperative footwear if walker boots or wounds were on the foot or ankle for foot injuries. Custom off-loading boots can be ordered at the discretion of the department investigator. In addition, the off-loading device used could be changed if necessary to accommodate changes in wound size or position.

Patients were evaluated weekly by the clinical department. The patient who achieved complete wound closure was then continuously assessed monthly during the follow-up period, twice for the first month and then for two subsequent visits. A control patient with a non-sutured wound to the end of the blinded treatment was able to receive the placental product of the open label therapy applied weekly until the placenta product (GRAFIX &lt; RTI ID = 0.0 &gt; (R) Results and safety assessments were made at each visit during blinded therapy, follow-up visit, as well as open-label treatment.

The primary endpoint of the study was complete wound closure evaluation of the index wound. Complete wound closure was defined as 100% re-epithelization without wound drainage as determined by department investigators. Confirmation of wound closure was made two weeks after the initial follow-up visit. The wound closure was independently verified through a central wound core laboratory with two blind wound care specialists who reviewed all scars via digitized acetic acid tracing and photographs. The secondary purpose included time for initial wound closure in patients receiving a control as measured by Kaplan-Meier analysis versus patients receiving placental products. A reduction of more than 50% of wound size by 28 days, the number of applications required for suturing, and the percentage of patients who achieved wound recurrence after initial wound healing were also determined. In open label therapy, wound closure by placental products was evaluated for patients in the control group in blinded therapy. The safety assessment included the number, type, and severity of adverse events as outlined in the National Cancer Institute's (NCI) CTCAE Version 3 standard for adverse events.

SAMPLE SIZE AND STATISTICAL ANALYSIS: The study sample size is based on an estimated closure rate (30% dropout rate) of 30% in the control arm and 50% in the placenta product group. Under these estimates, 94 complete patients in each treatment arm needed to meet both side 1 errors of 0.05 with 80% validity. A pre-specified interim analysis was planned at 50% registration. Intermediate analysis used a one-sided excellence design based on the Emerson-Fleming symmetric group using O'Brien-Fleming boundary geometry (Emerson and Fleming, 1989). Analysis was performed by a non-blinded statistician and reported to a blinded review committee. After the interim analysis, the blinded review committee recommended that the study registration be terminated because of the overwhelming superiority of the placenta product compared to the control arm.

Statistical analysis was performed using SAS version 9.2 for treatment intention analysis criteria. The baseline demographic and clinical variables were summarized for each treatment arm of the study. A descriptive summary of the distribution of continuous variables, including mean, standard deviation, median and target numbers, and categorical variables, is summarized for frequency and percentage. Treatment group summaries were constructed across all study divisions. Statistical comparisons between the treatment groups were performed using the Chi-square test for categorical variables and ANOVA for continuous measures. A Cox proportional hazard regression analysis was performed on the time vs. event (wound closure) data.

result

Patient demographics and baseline characteristics are presented in Table 16. During the screening, 139 patients were evaluated. Of the 42 patients who were not screened, 6 of them lost qualification after a week of running because they had a wound area reduction of 30% or more. 97 patients were subsequently randomized, 50 received placental products, and 47 received standard wound care. There was no significant difference in baseline characteristics between the two treatment groups. The planned interim analyzes showed overwhelming efficacy among patients receiving placental products for the primary and secondary endpoints compared to the control (Table 17). After the interim analysis, the Blind Review Committee recommended that the study registration be terminated due to overwhelming excellence.

Efficacy evaluation

The proportion of patients achieving a complete wound closure was 31% (50/50, 62.0%) compared to the control group (10/47, 21.0%, p = 0.0001) Was significantly higher in the patients who received it. The odds ratio for complete healing of placental product patients was 6.037 (95% CI 2.449-14.882) compared to the control. The placenta product group had a significantly faster median time for complete wound closure compared to the control group (42 vs. 69.5 days, p = 0.019) in wound in both sealed groups as shown in FIG. Kaplan-Meier analysis shows a statistically greater likelihood of complete wound healing during a 12 week evaluation period for placenta products (FIG. 34). The stitching probability for the placenta product group was 67.1% compared to 27.1% for the standard control group (log-rank method, p <0.0001). Patients with placenta products required less study visits (ie, application) to achieve suture than the control arm's patients (6 vs. 12, p <0.001), as shown in FIG. Comparisons of patients with at least a 50% reduction in wound size by day 28 showed that 31 (62.0%) of the 50 patients in the placenta product group achieved this reduction compared to 19 of the 47 (40.4%) in the control group (P = 0.035), respectively. Eight patients (16%) were missing from the study before the completion of the placenta product group compared to 11 patients (23.4%) who were excluded from the control group.

During the first 12 weeks of study, wound recurrence of the sealed DFU was evaluated. Follow-up at Week 2, Week 4, and Week 4 over a total of 12 weeks showed that ulcers were 82.1% (28 of 28 patients) of the placenta product group compared to 70.0% (7 of 10 patients) ) (P = 0.42).

Open markers : Patients in control arms that were not cured during the first 12 weeks of therapy could be crossovered to receive placental product therapy for up to 12 weeks (n = 26). After receiving placental product therapy, the stitching probability was 67.8% and the median time for stitching in these patients was 42 days. (Fig. 36)

Regression analysis: Cox proportional hazard regression analysis was performed using BMI as the treatment arm, duration of ulcer, baseline ulcer area, glucose control (glycated hemoglobin), ulcer location and covariate. After adjustment for these variables, placental products were found to have a significant effect on time for stitching (p < 0.0001). The hazard ratio was 4.77 (95% CI 2.279, 9.971), indicating excellent oz of suture by placental product for standard wound treatment.

Safety evaluation: Overall, placenta product patients who experienced at least one adverse events compared to the control patients was fewer (44.0% vs. 66.0%, p = 0.031). Among patients randomized to placental products, significantly fewer wound-related infections (18.0% versus 36.2%, p = 0.044), fewer serious adverse events associated with wound infections (8% versus 21.3% p = 0.084). There was less infection-related hospitalization in the placenta product group than in the control group (6% vs. 15%, p = 0.15). (Table 18).

Argument

Results demonstrate that weekly application of placental products as disclosed herein effectively improves the cure rate of diabetic foot ulcers and reduces diabetic foot complications compared to standard wound treatments. In this study, all primary and secondary endpoints presented clinical benefits of placental products only in multicenter DFU trials, which so far have met with statistically significant pre-specified interim analyzes. It is also the first report of a multicenter randomized controlled trial (RCT) to study human amniotic membrane for the treatment of diabetic foot ulcers. Additionally, the knowledge of the present inventors, it checked by a the first large, for the advanced skin substitute center RCT, wherein the first end point, 100% re-epithelialization was blinded third party (3 ^rd party) wound care professionals Thereby further eliminating the potential bias and increasing the reliability of the end point results. The placenta product group had a significantly higher complete seal rate (191% relative improvement) as shown in FIG.

In this study, surgical necrotic tissue removal was performed on all patients at study visit. Other studies have shown that a small number of DFUs have undergone surgical necrosis removal in three-phase DFU procedures (Steed, DL, et al., Diabetic Ulcer Study Group, J Am Coll Surg, 1996. 183 (1): 61-4; Saap, LJ and V. Falanga, Debridement performance index and its correlation with complete closure of diabetic foot ulcers Wound Repair Regen, 2002. 10 (6): p. 354-9) and reported that the frequency of wound necrosis is associated with wound healing. (See Steed et al., Supra, Table 19).

Example  23 Cryopreserved membranes in the tendon repair method

Keep the cryopreserved membrane at -80 ° C (± 5 ° C) until ready for transplantation. After thawing the frozen membrane, rinse using a protocol that provides a packaging insert. Additional information can be found in the Grafix Preparation Guide provided by Osiris Seraputics. After thawing, the rinsing step can be carried out and then the grafts can be held in the rinse head 1 hour prior to use. Once the membrane is thawed and rinsed, it is ready for transplantation.

Depending on whether the cryopreserved film has a directional (e.g., amniotic) membrane, the packaging can exhibit film orientation (e.g., the side is epithelium or unadhered side and / or the adhesive side of the substrate or membrane). When amniotic membrane is used, the substrate layer can be placed in contact with the restored tissue surface.

The following technique provides an example that is expected to be successfully demonstrated when using the cryopreserved membranes disclosed herein after tendon restoration covering.

Fabrication of membranes for application

Prior to the beginning of the application procedure, any perfusion of the wound and restoration site should be completed and any aspiration must be performed. This ensures that the membrane will not be trapped upon aspiration or destroyed after application. Any mounted support included with the cryopreserved membrane (e. G., Nitrocellulose) is carefully removed after thawing is complete. When provided with a film comprising amniotic membrane cryopreserved on a nitrocellulose support, the method of dispensing may comprise:

1. Using a 2-inch ribbon (with malleability), place the epithelial side of the membrane in contact with the ribbon. In this position, the substrate side of the membrane is exposed using a nitrocellulose frame on top of the membrane.

2. Using a single or two non-traumatic forceps, begin to slowly pull the nitrocellulose frame from the membrane while stabilizing the membrane (eg, using a finger). It may be desirable to always keep the membrane in contact with the ribbon to prevent bowling / shrinkage and to ensure proper identification of the substrate layer.

3. Once the thick plate is removed, use a forceps, finger, or freer elevator to carefully wrinkle the membrane. The membrane is now on the substrate layer, ready to be delivered to the tendon. See for example FIGS. 37A-B.

Delivery of membranes to tendons

After surgical repair of the tendons, the restrained tendon segments are carefully inserted to allow the ribbon to pass under the restoration site. The substrate side of the membrane will now touch the restored tendon. (Fig. 37C). Using a non-trauma forceps, carefully lift one entire side of the membrane and place it on the tendon segment. The membrane (adhesive side) will naturally bond to the tendons, but it can wrinkle and reposition if necessary. (Fig. 37D). While stabilizing the applied membrane with one of the forceps or other instruments, wrap the remaining membrane portion in the opposite direction to wrap the remaining tendon segment. Any extra film can be overlapped with previously applied film. (Fig. 37E). This application will completely enclose the restrained tendons in the barrier sheath. Because of the adhesion properties of the substrate layer and the potential inflammatory response from any reabsorbable sutures, such sutures may not be recommended for the graft to remain in place.

While the tendon is still in place, carefully remove the ribbon and reposition the tendon back into the sheath. Suture the tendon sheath carefully so as not to mix the membrane improperly. (Fig. 37F). If perfusion or aspiration is proven to be necessary, a sharp observation of the membrane will be essential to avoid membrane breakage or loss, or it may be advisable to carefully wipe with a Ray-tec sponge. Restrictions on the use of aspiration are expected to reduce the likelihood of membrane breakage.

Argument

Surgical reconstruction of tendon ruptures is common in most foot and ankle operative procedures. One of the most common postoperative complications is the adhesion of restoration tendons around soft tissue structures. Membranes containing the cryopreserved membranes disclosed herein, such as cryopreserved amniotic membranes, have been found to have naturally antiadhesive and anti-inflammatory properties. Therefore, it is ideally suited to use in a detrimental area where the resulting increase in inflammation and scar formation is. The membrane will not necessarily provide increased structural integrity and so the only use of the membrane in tendon rupture restoration is appropriate when structural reinforcement is not needed or when using the membrane in combination with other products that provide structural reinforcement. Recent studies have shown that when the cryopreserved membranes are used with an acellular dermis graft and PRP, the host inclusion rate of dermal grafts is improved. The research suggests a histological remodeling of the graft into the physiologically identical tissues of the graft site. Furthermore, increased incorporation rates, tissue differentiation and remodeling were observed. These features allow the use of cryopreserved membranes for inhibition / prevention of tissue adhesion, scar inhibition / prevention during postoperative period, and tendon restoration.

Example 23.1 A cryopreserved membrane in a tendon transfer method

A 72 - year - old female patient with painful nodule and chronic diverticulitis was admitted. The patient underwent surgery to remove the nodules (Fig. 38A). The cryopreserved membrane could be thawed as discussed above and was shaped like a needle (Fig. 38B). Subsequently, the membrane is inserted into the tendon, and then the tendon is sutured using a suture (Fig. 38C). The membrane is expected to promote accelerated wound healing and tissue repair as well as prevent adhesion to the restrained tendons.

references

Claims (79)

A film comprising a lyophilized amniotic membrane, said lyophilized membrane comprising, after lyophilization and subsequent thawing, said amniotic membrane comprising:
A) a tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells;
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) One or more types of depleted positive immunogenic cells. 2. The membrane of claim 1, wherein the type of depleted positive functional immunogenic cells is selected from maternal blood cells, neonatal blood cells, and tissue macrophages. 3. The method of claim 2, wherein the type of depleted positive functional immunogenic cell is a membrane comprising a cryopreserved amniotic membrane comprising at least one tissue macrophage selected from the group consisting of CD11b +, CD14 +, CD18 +, CD40 + and CD86 + . 4. The membrane of claim 1, wherein the depleted positive functional immunogenic cells produce an immunogenic factor in an amount less than a level sufficient to produce an immune response. 5. The membrane of claim 4, wherein the functional immunogenic cells produce TNF-a in an amount of less than 350 pg / cm2. 3. The membrane of claim 1, wherein the depleted positive functional immunogenic cells produce an immunogenic factor in an amount less than a detectable limit. The membrane of claim 1, wherein the extracellular matrix comprises at least one extracellular matrix protein. The membrane of claim 1, wherein said viable cell comprises at least one epithelial cell, hepatic stem cell, fibroblast, or a combination thereof. 2. The membrane of claim 1, wherein from about 50% to about 100% of the tissue cells are viable, cryopreserved amniotic membrane. 7. The membrane of claim 1, wherein from about 60% to about 100% of the tissue cells are viable, cryopreserved amniotic membrane. 7. The membrane of claim 1, wherein from about 70% to about 100% of the tissue cells are viable, cryopreserved amniotic membrane. The membrane of claim 1, wherein less than 10% of the viable cells are functional CD14 + macrophages, the membrane comprising a cryopreserved amniotic membrane. The membrane of claim 1, wherein less than 1% of the viable cells are functional CD14 + macrophages, the cryopreserved amniotic membrane. A film containing a lyophilized amniotic membrane, wherein, after lyophilization and subsequent thawing,
A) a substrate layer comprising a living cell, one or more therapeutic factors, and an extracellular matrix,
B) an epithelial layer comprising living cells and one or more therapeutic factors; And
C) one or more types of depleted amounts of functional immunogenic cells,
Wherein more than 40% of the combined cells in the substrate and epithelial layers are viable cells. A membrane comprising a cryopreserved placenta membrane, wherein after cryopreservation and subsequent thawing the placental membrane comprises a cryopreserved placental membrane comprising:
A) a tissue cell, said tissue cell being native to said placenta membrane and capable of surviving more than 40% of said tissue cells;
B) one or more therapeutic factors that are natural to the placental membrane;
C) extracellular matrix that is natural to the placenta; And
D) One or more types of depleted positive immunogenic cells. 16. The membrane according to claim 15, wherein the placental membrane comprises amniotic membrane and chorionic membrane. 16. The membrane of claim 15, wherein the type of depleted positive functional immunogenic cells is selected from maternal blood cells, neonatal blood cells, tissue macrophages, and trophoblasts. 18. The membrane of claim 17, wherein the depleted positive functional immunogenic cells comprise at least one tissue macrophage selected from the group consisting of CD11b +, CD14 +, CD18 +, CD40 + and CD86 +. 16. The membrane of claim 15, wherein the depleted positive functional immunogenic cells produce an immunogenic factor in an amount less than a level sufficient to produce an immune response. 20. The membrane of claim 19, wherein the at least one type of functional immunogenic cell produces TNF-a in an amount of less than 350 pg / cm2. 16. The membrane of claim 15, wherein the depleted positive functional immunogenic cell produces an immunogenic factor in an amount less than a detectable limit. 16. The membrane of claim 15, wherein the membrane is substantially depleted of placental nutrient, the cryopreserved placental membrane. 16. The membrane of claim 15, wherein said extracellular matrix comprises at least one extracellular matrix protein. 16. The membrane of claim 15, wherein the viable cell comprises at least one epithelial cell, hepatic stem cell, fibroblast, or a combination thereof. 16. The membrane of claim 15, wherein from about 50% to about 100% of the tissue cells are viable, the cryopreserved placental membrane. 16. The membrane of claim 15, wherein from about 60% to about 100% of the tissue cells are viable, the cryopreserved placental membrane. 16. The membrane of claim 15, wherein from about 70% to about 100% of the tissue cells are viable, the cryopreserved placental membrane. 16. The membrane of claim 15, wherein less than 10% of the viable cells are functional CD14 + macrophages, the cryopreserved placental membrane. 16. The membrane of claim 15, wherein less than 1% of the viable cells are functional CD14 + macrophages, the cryopreserved placental membrane. A film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing the at least one tissue component comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane; And
C) a natural extracellular matrix for said amniotic membrane;
The amniotic membrane has a depleted positive functional immunogenic cell type;
Wherein the at least one tissue component is present in an amount effective to provide a therapeutic benefit. A film comprising a cryopreserved amniotic membrane having at least one tissue component, wherein after cryopreservation and subsequent thawing the at least one tissue component comprises:
A) a tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells;
B) one or more therapeutic factors that are natural to said amniotic membrane; And
C) an extracellular matrix native to said amniotic membrane,
The amniotic membrane has a depleted positive functional immunogenic cell type;
Wherein the at least one tissue component comprises:
(i) reduce and / or reduce the amount and / or activity of proinflammatory cytokines;
(ii) increase the amount and / or activity of an anti-inflammatory cytokine;
(iii) reduce and / or reduce the amount and / or activity of reactive oxygen species;
(iv) increase the amount and / or activity of an antioxidant;
(v) reduce the amount and / or activity of the protease and /
(vi) increase cell proliferation and / or;
(vii) increase and / or increase neovascularization;
(viii) present in an amount effective to increase cell migration,
A film comprising a cryopreserved amniotic membrane having at least one tissue component. 32. The method of claim 31, wherein the at least one tissue component is administered in vitro:
(i) a decrease in the amount and / or activity of proinflammatory cytokines;
(ii) an increase in the amount and / or activity of anti-inflammatory cytokines;
(iii) reduction in the amount and / or activity of reactive oxygen species;
(iv) an increase in the amount and / or activity of an antioxidant;
(v) reducing the amount and / or activity of the protease;
(vi) increased cell proliferation;
(vii) increased angiogenesis; And / or
(viii) an amount effective to promote an increase in cell migration,
A film comprising a cryopreserved amniotic membrane having at least one tissue component. 32. The method of claim 31, wherein the at least one tissue component is administered in vivo:
(i) a decrease in the amount and / or activity of proinflammatory cytokines;
(ii) an increase in the amount and / or activity of anti-inflammatory cytokines;
(iii) reduction in the amount and / or activity of reactive oxygen species;
(iv) an increase in the amount and / or activity of an antioxidant;
(v) reducing the amount and / or activity of the protease;
(vi) increased cell proliferation;
(vii) increased angiogenesis; And / or
(viii) an amount effective to promote an increase in cell migration,
A film comprising a cryopreserved amniotic membrane having at least one tissue component. 31. The method of claim 30, wherein when the membrane is applied to a subject in need of a therapeutic benefit,
(i) reduce and / or reduce the amount and / or activity of proinflammatory cytokines;
(ii) increase the amount and / or activity of an anti-inflammatory cytokine;
(iii) reduce and / or reduce the amount and / or activity of reactive oxygen species;
(iv) increase the amount and / or activity of an antioxidant;
(v) decreasing the amount and / or activity of the protease;
(vi) increase cell proliferation and / or;
(vii) increase and / or increase neovascularization;
(viii) present in an amount effective to increase cell migration,
A film comprising a cryopreserved amniotic membrane having at least one tissue component. 35. The membrane according to any one of claims 1 to 34, wherein the amniotic membrane is fixed to a delivery substrate. 36. The membrane of claim 35, wherein the delivery substrate is nitrocellulose. 35. The membrane of any one of claims 1 to 34, wherein the cryopreserved amniotic membrane is stored for a prolonged period of time prior to subsequent thawing. 38. The membrane of claim 37, wherein the prolonged period of time is from about 6 months to at least about 36 months. 38. The membrane of claim 37, wherein the viability of the tissue cells is substantially maintained during thawing. 35. The membrane of any one of claims 1 to 34, wherein said viability of said tissue cells is substantially maintained for at least about 25 months when cryopreserved. 35. The membrane according to any one of claims 1 to 34, wherein the cryopreserved amniotic membrane is thawed and can be ready for use within the first 30 minutes of the thawing method. A method of treating a wound on a subject comprising administering a film comprising amniotic membrane cryopreserved at the wound site, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) at least one type of depleted amount of functional immunogenic cells;
Upon administration, the membrane provides the living cell, extracellular matrix and / or one or more therapeutic factors in an amount effective to promote one or more of the following:
(i) a decrease in the amount and / or activity of proinflammatory cytokines;
(ii) an increase in the amount and / or activity of anti-inflammatory cytokines;
(iii) reduction in the amount and / or activity of reactive oxygen species;
(iv) an increase in the amount and / or activity of an antioxidant;
(v) reducing the amount and / or activity of the protease;
(vi) increased cell proliferation;
(vii) increased angiogenesis; And / or
(viii) Increased cell migration. 43. The method of claim 42 wherein said wound is selected from the group consisting of lacerations, abrasions, burns, wounds, holes caused by projectiles, epidermal wounds, skin wounds, chronic wounds, acute wounds, external wounds, internal wounds, Selected from the group consisting of diabetic ulcers, wounds caused in addition to surgical procedures or in addition to surgical procedures, vein skin ulcers, spinal cord injuries, eye wounds, eye injuries, ear injuries, otitis media wounds or injuries, eye injuries and avascular necrosis , A method of treating wounds. 43. The method of claim 42, wherein the wound is selected from the group consisting of lacerations, burns, chronic wounds, acute wounds, diabetic ulcers, wounds caused by or in addition to surgical procedures, and vein skin ulcers. 43. The method of claim 42, wherein the membrane promotes, directly or indirectly, one or more of the following:
(i) a decrease in the amount and / or activity of proinflammatory cytokines;
(ii) an increase in the amount and / or activity of anti-inflammatory cytokines;
(iii) reduction in the amount and / or activity of reactive oxygen species;
(iv) an increase in the amount and / or activity of an antioxidant;
(v) reducing the amount and / or activity of the protease;
(vi) increased cell proliferation;
(vii) increased angiogenesis; And / or
(viii) Increased cell migration. A method of treating or preventing tissue adhesion associated with a surgical procedure comprising administering a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing,
A) tissue cells, tissue cells which are native to the amniotic membrane and in which more than 40% of the tissue cells are viable;
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) a depleted amount of one or more types of functional immunogenic cells,
Wherein the membrane is administered to a tissue and provides a survival cell, an extracellular matrix and / or one or more therapeutic factors in an amount effective to treat or prevent tissue adhesion. A method of accelerating wound healing in a subject having a wound requiring healing,
a) administering to the wound site a membrane according to any one of claims 1 to 34,
Wherein the administering step is effective to promote wound closure up to 12 weeks after the initial administration step. A method of accelerating wound healing in a subject having a wound requiring healing,
a) administering to the wound site a membrane according to any one of claims 1 to 34,
Wherein the administering step is effective to promote wound closure for 5 to 6 weeks after the initial administration step. A method of accelerating wound healing in a subject having a wound requiring healing,
a) administering to the wound site a membrane according to any one of claims 1 to 34,
Wherein the administering step is effective to promote a reduction in wound size by at least 50% by 28 days after the initial administration step. A method of improving wound closure rate in a subject having a wound requiring healing,
comprising the steps of: a) administering to the wound site a membrane according to any one of claims 1 to 34,
Wherein said administering is effective to improve wound closure rate by at least about 44% relative to standard wound treatment. CLAIMS What is claimed is: 1. A method of treating a subject for a wound that is refractory prior to a wound healing treatment,
a) administering to the wound site a membrane according to any one of claims 1 to 34,
Wherein said administering is effective to promote wound closure up to 12 weeks after the initial administration step. A method of treating chronic wounds comprising administering a film comprising amniotic membrane cryopreserved to the wound site, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) at least one type of depleted amount of functional immunogenic cells,
Wherein said administering provides a viable therapeutic cell, an extracellular matrix and one or more therapeutic factors in an amount effective to promote healing of chronic wounds. CLAIMS What is claimed is: 1. A method of treating inflammatory eye disease in a subject comprising administering to the subject a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing,
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
Wherein said administration provides a survival therapeutic cell, an extracellular matrix and one or more therapeutic factors in an amount effective to treat inflammatory eye diseases. 57. The method of claim 53, wherein the inflammatory eye disease is a wound or disease characterized by inflammation. A method of promoting tissue repair and / or tissue regeneration in a subject comprising administering to the subject a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) at least one type of depleted amount of functional immunogenic cells,
Wherein said administration provides a survival therapeutic cell, an extracellular matrix and one or more therapeutic factors in an amount effective to promote tissue repair and / or tissue regeneration. 57. The method of claim 55, wherein the method is used in conjunction with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal cord surgery to promote tissue repair and / Way. 57. The method of claim 55, wherein the tissue is human tissue, wherein the tissue repair and / or tissue regeneration is promoted in the subject. 57. The method of claim 55, wherein said human tissue is cartilage, skin, ligament, tendon or bone, which promotes tissue repair and / or tissue regeneration in a subject. 56. The method of claim 55, wherein the membrane stimulates tissue regeneration, directly or indirectly, to promote tissue regeneration and / or tissue regeneration in a subject. CLAIMS What is claimed is: 1. A method of modulating an inflammatory response comprising administering a film comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) at least one type of depleted amount of functional immunogenic cells,
Wherein said administration modulates an inflammatory response that provides said viable cell, extracellular matrix, and / or one or more therapeutic factors in an amount effective to reduce the amount of pro-inflammatory cytokine and / or increase the amount of anti-inflammatory cytokine How to. 61. The method of claim 60, wherein said proinflammatory cytokine consists essentially of TNF- [alpha] and IL-l [alpha], or a combination thereof. 61. The method of claim 60, wherein said anti-inflammatory cytokine consists essentially of IL-10 or PGE2 or a combination thereof. A method of modulating protease activity comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
Wherein said administration comprises administering a therapeutically effective amount of a protease inhibitor, said protease inhibitor, which provides said living cell, extracellular matrix, and / or one or more therapeutic factors in an amount effective to reduce and / or increase the amount and / or activity of the protease or to increase the amount and / Lt; / RTI &gt; 65. The method of claim 63, wherein the protease is selected from the group consisting of a substrate metalloproteinase (MMP) and an elastase, or a combination thereof. 66. The method of claim 63, wherein said at least one protease inhibitor comprises a tissue inhibitor of matrix metalloproteinase (TIMP). A method of reducing the amount and / or activity of a reactive oxygen species (ROS) comprising administering a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
The administration can be carried out in the presence of a reactive oxygen species which provides a viable cell, an extracellular matrix and / or one or more therapeutic factors in an amount effective to reduce the amount and / or activity of ROS and / or increase the amount and / Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; 67. The method of claim 66, wherein said surviving therapeutic cells, extracellular matrix and one or more therapeutic factors are selected from the group consisting of a reduced amount and / or activity of reactive oxygen species, provided in an amount effective to provide the same antioxidant capacity as a 250 mM solution of ascorbic acid. How to do it. CLAIMS What is claimed is: 1. A method of increasing angiogenesis comprising administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) a tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells;
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
Wherein said administration provides said viable cell, extracellular matrix, and / or one or more therapeutic factors in an amount effective to increase angiogenesis. 69. The method of claim 68, wherein said surviving treatment cells, extracellular matrix and one or more therapeutic factors are selected from the group consisting of vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) / RTI &gt; and / or &lt; RTI ID = 0.0 &gt; activity. &Lt; / RTI &gt; A method of increasing cell migration to a wound site comprising administering a film comprising amniotic membrane cryopreserved to the wound, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
Wherein said administration provides said viable cell, extracellular matrix, and / or one or more therapeutic factors in an amount effective to increase cell migration. 71. The method of claim 70, wherein said cell migration comprises cells selected from the group consisting of endothelial cells, fibroblasts and epithelial cells, and combinations thereof. CLAIMS What is claimed is: 1. A method of increasing cell proliferation, comprising: administering a membrane comprising amniotic membrane cryopreserved to a wound, wherein after cryopreservation and subsequent thawing,
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) comprises at least one type of depleted amount of functional immunogenic cells;
Wherein said administration provides said viable cell, extracellular matrix, and / or one or more therapeutic factors in an amount effective to increase cell proliferation. CLAIMS What is claimed is: 1. A method for preventing or reducing scarring or build-up in a subject comprising administering to said subject a film comprising a cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing,
A) tissue cell, said tissue cell being native to said amniotic membrane and capable of surviving more than 40% of said tissue cells
B) one or more therapeutic factors that are natural to said amniotic membrane;
C) an extracellular matrix that is natural to the amniotic membrane; And
D) depleted positive functional CD14 + macrophages;
Wherein said administering provides a survival therapeutic cell, an extracellular matrix and one or more therapeutic factors in an amount effective to prevent or reduce scarring or build-up, a method of preventing or reducing scarring or build-up formation. 73. The method of claim 73, wherein said surviving therapeutic cells, extracellular matrix and one or more therapeutic factors are selected from the group consisting of: preventing scarring or build-up in a subject that is present in an amount effective to increase IFN-2 alpha in an amount effective to prevent or reduce scar formation; / RTI &gt; 74. The method of claim 74, wherein said surviving therapeutic cells, extracellular matrix and one or more therapeutic factors are selected from the group consisting of: preventing scarring or build-up in a subject that is present in an amount effective to increase TGF-? 3 in an amount effective to prevent or reduce scar formation; / RTI &gt; 76. The method according to any one of claims 59 to 75, wherein administration of the membrane to the wound stimulates or induces the method directly or indirectly. A kit for the treatment of a wound or tissue defect comprising:
A) a membrane according to any one of claims 1 to 34 in a pharmaceutically acceptable container; And
B) Instructions for administering the membrane to treat the wound or tissue defect. 77. The kit of claim 77, wherein the kit further comprises an adduct. 79. The composition of claim 78, wherein the adduct is one or more of an antibiotic, an emollient, a keratolytic agent, a wetting agent, an antioxidant, a preservative, a therapeutic, a bandage, a tool, a cutting device, a buffer, a thawing medium, A kit for the treatment of wounds or tissue defects, selected from water.

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