WO2015134946A1 - Amnion derived therapeutic compositions and methods of use - Google Patents

Abstract

Therapeutic composites are described for the treatment of a variety of organ related conditions. Any organ or organ system of the body may be treated including the pancreas, kidney, brain, lung, intestine and the like. A therapeutic composition may include a fluid component and a matrix component. A matrix component may be an amniotic membrane and a fluid component may be a fluid having a concentration of amniotic stem cells, and/or micronized amniotic membrane particles. An amniotic stem cell concentrated fluid has at least 0.1 x 10e amniotic stem cells per milliliter of fluid or composition. A therapeutic composition may be used to treat any number of conditions through topical application, injection, or intravenously. A matrix component may be located on a treatment location and a fluid component may subsequently be introduced to the matrix component or the surrounding tissue.

Description

AMNION DERIVED THERAPEUTIC COMPOSITIONS AND METHODS OF USE

BACKGROUND OF THE INVENTION

Cross Reference To Related Applications

[0001] This application claims the benefit of U.S. provisional patent application no. 61/949,066 , to Amnio Cordis Technology LLC, U.S. provisional patent application no. 61/949,135, to Amnio Cordis Technology LLC, provisional patent application no, 61/949,106, to Amnio Technology LLC and U.S. provisional patent applicatio no. 61/949,087 to Amnio Technology, all filed on March 6, 2014; the entirety of each application is incorporated herein by reference.

Field of the invention

[0002] The present invention relates to therapeutic compositions derived from amnion materials and methods of use.

Background

[0003] Amniotic membranes are being used in clinical trials to treat a wide range of conditions. Amniotic membranes are typically placed directly on a treatment location, such as a wound or scare. In many cases however, amniotic membranes Sack the proper architecture and cell viability to effectively provide the desired therapeutic responses, such as tissue regenerations, i munomoduSation, anti-inflammatory and antiftbrotic. Most amniotic membranes are dehydrated and cryogenicaiiy preserved. In other cases, the amniotic membranes are sterilized in a manner that damages the tissue and/or reduces cell viability. For example, many amniotic membranes are processed with a glutaraSdehyde which is known to significantly reduce cell viability. In many treatment applications, it is desirable to provide a high concentration and/or specific type or b!end of stem ceils, in addition, some therapeutic composites comprise components from two or more donors thereby limiting their use.

SUMMARY OF THE INVENTION

i £0004] The invention is directed to therapeutic compositions that, in one embodiment, comprise a therapeutic fluid comprising amniotic fluid. An amniotic fluid may comprises any number of cells, including stem ce!!s, growth factors, proteins and the like, in one embodiment, a therapeutic fluid comprises an amniotic fluid that is aceiiuiar. In another embodiment, a therapeutic composition comprises a matrix component, such as an amniotic membrane. In still another embodiment, a therapeutic composition comprises a matrix component and a fluid component, wherein a fluid component may be imbibed into o coated onto one or more surfaces of the matrix component. In an exemplary embodiment, a therapeutic composition comprises an amniotic membrane in the matrix component and comprises amniotic fluid in the fluid component,

[0005] in an exemplary embodiment, the therapeutic composition, as described herein, comprises a plurality of amniotic stem cells, and preferably at a high concentration, such as greater than 0.5 x 10^s per milliliter of the therapeutic fluid component within the therapeutic composition. A therapeutic fluid component may also be aceSlular, such as an acetluiar amniotic fluid. An aceliu!ar amniotic fluid is described in U.S. application no 14/593,415 to Amnio Technology LLC; the entirety of which is incorporated by reference herein, A therapeutic fluid component may be referred to herein as simply a fluid component for brevity. In another embodiment, a fluid component comprises amniotic membrane that has been micronized and dispersed in a fluid. In one embodiment, a fluid component Is a dispersion of micronized amniotic membrane combined with a fluid, such as plasma, saline, amniotic fluid, combinations thereof and the like, in another embodiment, a fluid component comprises a mixture of micronized amniotic membrane particles combined with an amniotic stem eel! concentrated fluid, in still another embodiment, a therapeutic fluid consists essentially of a concentrated amniotic fluid wherein the quantity of amniotic stem cells is increased. The amniotic stems cells in the therapeutic composite, as described herein, may be derived from amniotic fluid and the stem cells may be concentrated by a centrifuge process. Additional fluids and agents may be added to the amniotic stem ceils such as plasma, Plasma Lyte-A, from Baxter Inc., saline and the like. The concentration of amniotic stems ceiis In one milliliter of a fluid component of an exemplary therapeutic composition, as described herein, may be about 0.5 x 10^a or more, 1.0 x 10⁹ or more, 5.0 x 1Q^ft or more, 10 x 10^e or more and any range between and including the concentrations values provided. A high concentration of amniotic stems cells may greatiy improve the effectiveness of the therapeutic composition for many applications. The therapeutic composition, as described herein, may comprise endothelial cells, mesenchymal stem cells, amniotic fluid stem ceils, fibroblasts, proteins,

keritinocytes, epithelial and/or epidermal celis, parafenacyfes, keratinoeytes, epithelial and/or epidermal cells, paratenacytes, keratinoeytes and growth factors. In some embodiments, protein markers for mesenchymal stem cells may be analyzed to quantify the various types of ceils within the therapeutic composition. Flow cytometry may be used to identify proteins, CD44, CD 105. CD73 and CD90. In one embodiment, a therapeutic composition comprises at least 30% of mesenchymal stem cells as identified by CD73. Mesenchymal stem cells indicated by CD73 proteins may be more mobile and provide a more therapeutic effect that

mesenchymal stem cells identified by the other markers. A therapeutic fluid component, as described herein, may comprise anti-inflammatory nano-particles and/or statins, Mfv!G-CoA reductase inhibitors to reduce inflation at a treatment location.

[0006] In some embodiments, a therapeutic composition is doped with progenitor cells and the progenitor cells may be multipotent progenitor cells and/or piuripotent progenitor ceils. Progenitor celis may be derived from a patient to be treated, such as from a stromal vascular fraction. Vascular fraction cells and/or progenitor celis may be included with a therapeutic composite to further improve effectiveness. Progenitor cells may be autologous or allogeneic.

[0007] A fluid component, as described herein, may comprise particles and/or a concentration of amniotic stem cells. The particles within the fluid component may comprise micronized amniotic membrane. The micronized amniotic membrane may comprise hydrated mammalian amniotic tissue having a percent hydration of at least about 25%, at least about 50%, at least about 75% by weight or any range between the concentrations provided. Amniotic membrane maintained in a hydrated state may provide for more viable and regenerative properties, Amniotic membranes that are Syophilized have a great reduction in ceil viability. The particles in the fluid component, as described herein, may consists essentially of amniotic membrane and be substantially free of chorion. The amnion layer may be removed from the chorion prior to processing. In one embodiment, the amniotic membrane particles consist essentially of epithelium wherein the concentration of the epithelium is about 70% or more, for example. The particies consisting essentially of epithelium may comprise stem ceils and tissue that may substantially surround the stem cells,

[0008] An amniotic membrane, or portion thereof, may be mlcronized while in a hydrated state thereby improving the viabilit of cells. The amniotic membrane particies may be derived from dehydrated and/or deceilularized amniotic tissue however. In addition, fhe amniotic membrane may be ciyo-fractured, such as with a blunt object to minimize shear and damage to tissue, thereby improving therapeutic effectiveness. Particies of amniotic membrane may have an suitable particle size, average particle size and particle sized distribution. For example, the amniotic membrane derived particles, or micronized particles, may have a particle size, or an average particle size of no more than about 10pm, no more than about 5pm, no more than about 2pm. no more than about 1 pm, no more than about 0.5pm and any range between and including the average particle sizes provided. The particle size of the amniotic membrane particies can be determine through any suitable method, including image analysis, whereby a therapeutic composite is dried and imaged using a scanning electron micrograph (SEM). The amniotic membrane derived particies may have an irregular shape and in some embodiments are planar having a first pianar surface and a second planar surface. Cryo-fracturing of amniotic membrane with a blunt object provides particies with less shear and a more irregular shape than conventional cryo-milling, thereby providing a higher surface area and more effective therapeutic effect.

[0009] The concentration of particles, such as micronized amniotic membrane, in the therapeutic composition and/or fluid component may be provided in any effective amount such as more than about 0.1 %, more than about 0.5%, more than about 1 %, more than about 10%, more than about 25%, more than about 50%, more than about 75%,or more than about 90% by weight of therapeutic composition and any range between and including the weight percentages listed. Likewise, the mass of particles, suc as amniotic membrane particies, may be provided in a therapeutic fluid component of a therapeutic composition in any effective amount, such as more than about I mg/mi, more than about 5mg/ml, more than about 10mg/ml, more than about 50mg/ml, more than about lOOmg/ml, more than about 500mg/ml, and any range between and inciuding the mass concentrations provided. The particies in the therapeutic composition may comprise collagen, growth factors, stem cells, amniotic stem ceils, mesenchymal stem ceils, progenitor cells, red b!ood ceils, white blood cells, proteins, fibroblasts, paratenacytes, keratinocytes and the like.

[0010] An exemplary therapeutic composition may comprise an oxygen-carrier component that may increase the effectiveness of the therapeutic composite by increasing oxygen availability and increase stem cell viability. Any suitable oxygen- carrier component or combination of components may be included into a therapeutic compositing including, but not limited to, perf!uorocarbon such as

perfluorotributySamine (PFTBA), perfluorooctySbromide {PFOB},

perfSuorodecylbrornide, perfluoroperhydrophenanthrene and the like. An oxygen- carrier may be bonded, such as covalently bonded to a therapeutic composition, such as to a matrix component or to the micronized amniotic membrane. In one embodiment, a matrix component comprises a polymeric material, such a

fiuoropoSymer, and an oxygen component is bonded thereto. Any suitable means may be used to bond an oxygen component to a therapeutic composition component including, cross-linking agents, radiation, and the like, in still another embodiment, an oxygen-carrier component may form a emulsion, or micro-emulsion with another fluid component. A peril uorocarbon oxygen-carrier component is hydrophobic and when mixed with a fluid componeni that is hydrophilic or comprises water, a emulsion may be formed comprising an aqueous phase and a perfl uorocarbon phase.

[001 1 ] Any of the fluid components described herein may be an injectable solution that will pass through a 20 gauge needle or a needle having a smaller diameter. In other embodiments, a fluid component is provided in a thicker composition, such as a paste that may be applied topically. The viscosity of the an injectable fluid component may be no more than about 1 mPa sec, no more than about 500 mPa sec, no more than about 1000 mPa sec, no more than 20,000 mPa sec, no more than 50,000 mPa sec. In other embodiments, a fluid component may be provided for topical applications and the viscosity may be more than about 20 Pa sec, more than about 50 Pa sec, more than about 100 Pa sec, more than about 250 Pa sec and any range between and including the viscosity values provided.

[0012] In an exemplary embodiment, a therapeutic composition is a

therapeutic composite and comprises any of the fluid components, as described herein, imbibed into or coated onto a matrix component. A matrix component is a sheet, block, tube or rod of material, for example, that may comprises porosity and pores for accepting a fluid component therein. A matrix component may be a biological material such as an amniotic membrane, in another embodiment, an amniotic membrane may be provided as a matrix component in a multiSayered configuration or combined with any other suitable support iayer for a desired

application. For example, a therapeutic composite, as described herein, may comprise an amniotic membrane layer and a cover layer. A cove Iayer may be used to reduce the loss, wash-out, of a fluid component from the therapeutic composite. In another embodiment, the therapeutic composite comprises an amniotic membrane and a support layer, such as a polymer matrix materia! including, but not limited to, a bioresorbable or fluoropolymer membrane. A support iayer may have a tensile break strength that is much greater, such as two times or more that of an amniotic membrane Iayer in a matrix component. In still another embodiment, a therapeutic composite comprises one or more layers of amniotic membrane that are tensilized, whereby an amniotic membrane has been stretched in one or more directions to increase strength and/or area of the membrane. An amniotic membrane may be cross-linked, and a cross-linked amniotic membrane may be combined with a non- cross-linked amniotic membrane. Any suitable method, as known in the art of cross- linking an amniotic membrane may be used including chemical, such as treatment with glutaraidebyde, radiation and the like. A therapeutic composite as described herein, may comprise anti-inflammatory nano-partic!es a d/or statins, H G-CoA reductase inhibitors to reduce inflation at a treatment location. An exemplary therapeutic composition, and in particular a fluid component, may comprise mannitol, saline, ringers lactate, vitamin 8 complex and the like.

[0013] A therapeutic composite, as described herein, may be provided with the therapeutic fluid imbibed into, coated onto, or otherwise applied to a matrix component. For example, a therapeutic composite comprising an amniotic membrane may be provided with a therapeutic fluid component comprising micronized amniotic membrane particles dispersed in fluid component. This carrier fiuid may be an amniotic stem ceil concentrated fluid component. In an exemplary embodiment, the therapeutic fluid component and the amniotic membrane are from a single donor. In an exemplary embodiment, the amniotic membrane, the micronized amniotic membrane particles and the amniotic stem cells in the fluid component are ail from a single donor. In another exemplary embodiment, a therapeutic composite comprises an amniotic membrane Iayer configured for direct application to a treatment location, a cover layer of a bioresorbable material and a therapeutic fluid component comprising a high concentration of amniotic stem cells. A portion of a bioresorbable materia! or other matrix layer of the therapeutic composite may be porous to enable a portion of the fluid component to be retained therein. Any suitable number and type of matrix or support layers may be configured in a therapeutic composite, as described herein. In one embodiment, a fluid component may be vacuum imbibed into a matrix component. Whereby a matrix component is submerged in a fluid component and vacuum is applied to remove substantially all the air from the matrix component. This removal of air will allow the fluid component to more substantially fill the voids and porosity of the matrix component.

[0014] A support layer may comprise any suitable type of material including, but not limited to, a bioresorbable material, a non-bsoresorbable polymer material, such a polyether ether keton (PEEK), or polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), perfiuoroa!koxy (PFA) and the like, or a metallic component, such as stainless steel, titanium, gold and the like, A support layer may be porous and/or permeable. A support iayer may be a membrane having a microstructure of pores, or a film, net, screen, woven and the like. A support Iayer may be substantially non-permeable to fluid and may be hydrophobic or oleophobsc on at least one side, in an exemplary embodiment, a support layer is expanded PTFE, In an exemplary embodiment, a support iayer is a sheet of material having a first substantially planar surface, a second substantially planar surface and a thickness.

[0015] A therapeutic composition may be introduced to a treatment location by direct topical application, such as b coating, applying, spraying, or placing over a treatment location and in some cases adhering a portion of the therapeutic composition with an adhesive, staples or sutures. In other embodiments, a

therapeutic composition is delivered transcathete and may be configured on any suitable implantable or delivery device, such as a stent or a deployabSe and removable balloon. In some embodiments, the fluid component, as described herein, is applied to the treatment location with both the matrix component and fluid component combined in a single step, whereby the fluid component is imbibed, coated or otherwise combined with the matrix component. In other embodiments, a matrix component is applied to a treatment location and a fluid component is subsequently added, such as by injection or topical application. For example, an amniotic membrane may be applied to a treatment location and a fluid component may subsequently be injected into the amniotic membrane and/or to the tissue under or around the location of the amniotic membrane. In still another embodiment, a first matrix component layer may be located on a treatment location and a second matrix component layer may be applied over the first matrix component layer. The first and/or second layer may comprise a fluid component and each layer may comprise a different composition of fluid component. A second matrix component layer may be substantially non-permeable to the fluid component thereby reducing wash-out or dilution of the fluid component from bodiiy fluid exposure.

[0016] A therapeutic composition, as described herein, comprises other biological materials that are not amnion derived. In one embodiment, a therapeutic composition comprises a stromal vascular fraction (SVF) from a patient that is to be treated with the therapeutic composition. Stromal vascular fraction derived from adipose tissue of a patient, for example, may be combined with the matrix and/or fluid component as described herein. In an exemplary embodiment, stromal vascular fraction is combined with micronized amniotic membrane and/or amniotic stem cells to form a fluid component. In another embodiment, a stroma! vascular fraction is combined with a matrix component either before or after locating the matrix component over the treatment location. The stromal vascular fraction may contain any of the following: preadipocytes, mesenchymal stem cells (MSG), endothelial progenitor ceils, T cells, B cells and mast cells as well as adipose tissue macrophages. In another embodiment, a therapeutic composition comprises bone marrow aspirate (BMA) and/or platelet rich plasma (PRP).

[0017] The therapeutic composition, as described herein, may be

cryopreserved whereby the temperature of the therapeutic composite is lowered to a temperature of no more than -70°C, and preferable lower than about -80°C. The rate of cooling may be controlled to reduce damage and maintain viability of the cells upon thawing.

[001 S] As shown in Table 1 below, an exemplary fluid component comprising a concentrated amniotic stem ceil fluid and micronized amniotic membrane particles, as described in Example 1 , retained a very high viability post controlled rate freezing. Maintaining the amniotic membrane in a hydrated state prior to cryo-fracturing and subsequent cryopreservtng improves cell viability. Tab!© 1 :

[0019] The viability of cel!s was maintained after thawing a cryo-preserved concentrated amniotic fluid as reported in Tabie 1. A small ioss in viability was observed with a totai viability after thawing a cryopreserved therapeutic composit of more than 90% in all cases. A therapeutic composite, as described herein, may have a cell viability of about 70% or more, at least about 80% or more, about 90% or more and an range between and including the ceil viability values provided.

[0020] Any of the therapeutic compositions described herein may be used for a wide variety of treatment applications. A therapeutic compositions, as described herein, may be provided to any suitable treatment location of the body to induce an immunomodulatory and/or anti-inflammatory response. In another application, a therapeutic composition is introduced into a treatment location to reduce scaring and to promote healing, whereby the therapeutic composition aids in regeneration of new tissue, A fluid component of the therapeutic composition, as described herein, may be injected directly into an affected area or introduced intravenously. It may be desirable to provide a fluid component comprising both amniotic stem cells and micronized amniotic membrane when tissue regeneration is desired. The micronized amniotic membrane particles may provide the architecture needed for more effective regeneration and tissue repair. j 0021 ] A therapeutic composition, as described herein, may be introduced into any organ of the body including, but not limited to. the heart, brain, Sung, liver, kidney, pancreas, stomach, intestine and the like through transcatheter, direct injection, or topicai application, in some embodiment, a therapeutic composition, as described herein is configured onto a stent or is positioned by a balloon catheter. In another embodiment, a therapeutic composition comprising a matrix component is configured onto a treatment location and in some cases may be wrapped around a tubular anatomical body portion, such as an intestine, ureter, urethra, faliopian tubes, vas derrens and the like, A matrix component may be wrapped around the tubular body part during a surgical procedure or through the use of orthoscopsc procedures.

[0022] A therapeutic composition, as described herein, may be used to treat urology conditions including post-operative scarring and strictures, for example. Strictures in the ureter and urethra may be treated by placement of a therapeutic composite to a stricture location to reduce scarring and blockage of the ureter.

Stricture treatment may comprise configuring a therapeutic composite around at least a portion of the ureter or urethra. In some embodiments, a therapeutic composite is wrapped completely around a ureter or urethra.

[0023] A therapeutic fluid, as described herein, may be provided intravenously to regulate systemic immun modulation post-surgery. An exemplary therapeutic fluid may comprise mannitoi, saline, ringers lactate, vitamin B complex and the like.

Mannitoi may be incorporated into therapeutic compositions for introduction into the brain, as the mannitoi may heip the treatment composition from passing the into the brain through the brain treatments

[0024] A therapeutic composition may be used to treat any number of lung related conditions, including, but not limited to, lung fibrosis, chronic obstaictive pulmonary disease (COPD), acute lung injury, transplanted lung rejection, puimonary hypertension, ventilator induced iung injury, acute respiratory disease syndrome (ARDS), bronchitis, alveolitis, chronic parenchymal and pleura! lung disease, trans- bronchial parenchymal disease, post-operative lung recovery and scaring and/or empyema. Treatment of these lung conditions may include introduction of a therapeutic composite, as described herein, to a iung by topical application to a portion of a iung, inter-arteriaily, intravenously into the venous portion of the circulatory system including the pulmonary artery, through a centra! venous catheter, peripheral venous catheter and the like. [0025] A therapeutic composite may be used to treat any number of brain related conditions, including, but not limited to, stroke, ischemic stroke, traumatic brain injury and post-operative brain recovery, g!iobastoma, and scaring. Additional brain related conditions thai may be treated with a therapeutic composition, as described herein include, but are not limited to, Parkinsons, autism, CVA's, TiA's depression, macular degeneration, dementia, neurofibromatosis, neuro

degenerative diseases, charcot-marie-tooth, Multiple sclerosis, and other neuro degenerative diseases i.e. ALS and the like. Treatment of these brain conditions may include introduction of a therapeutic composite, as described herein, by placement during surgery to an affected area of the brain. In another embodiment, a

therapeutic fluid is provided to a portion of the brain through IV infusion, intra arterial infusion, through a shunt or any suitable port, in order to enable or increase the passage of the therapeutic fluid to the brain, mannitol may be incorporated into the fluid, along with saline in some cases.

[0026] A therapeutic composite may be used to treat any number of other organ related conditions including, but not limited to, diabetes, renal failure, kidney failure, post-operative kidney or liver recovery and scaring, and the like. Treatment of these conditions may include introduction of a therapeutic composite, as described herein, topically, by transcaiheter and the like. An effective does of fluid component may be provided in one treatment or in several doses over a period of time. The specific treatment and dosing regime will depend on the type and severity of the condition to be treated,

[0027] A therapeutic composition comprising a therapeutic matrix component ma be applied to any organ, or tissue for adhesion prevention. For example, post an anastomosis or other operative procedure of a ureter, urethra, fallopian tubes, intestine or vas deferens, a matrix component may be applied over an effected area with or without a fluid component. Any anatomical body portion that can be constricted may have a matrix component, as described herein, wrapped around for the purpose of treatment after surgery, for example.

[0028] A therapeutic composition may be used to treat erectile dysfunction through injection of a therapeutic fluid into and around the base of the penis, such as in and around the neurovascular bundles to reduce vascular constriction. In addition, patients suffering from Peyronie's disease may be treated by injection to the affected area of the penis. [0029] In one embodiment, a fluid component is injected into a specific treatment location through the use of a catheter, such as a steerabie catheter and an injection imptement configured on the introductory end of the catheter. For example, a catheter having an injection implement may be introduce into the femora! artery, inserted to position the injection implement in proximity to an organ, whereby a dose of therapeutic composite is administered into the organ.

[0030] The summary of the invention is provided as a genera! introduction to some of the embodiments of the invention, and is not intended to be limiting.

Additional example embodiments including variations and alternative configurations of the invention are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031 ] The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention, and together with the description serve to explain the principles of the invention.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[0032] Figure 1A shows a cross-sectiona! diagram of amniotic membrane surrounding a fetus in utero.

[0033] Figure 18 shows a cross-section diagram of the layers of the amnion and chorion.

[0034] Figure 2A show a transmission electron micrograph (TEM) of the epithelium layer of the amniotic membrane having a single iayer of amniotic stem cells. The TEM is at 2600 X magnification.

[0035] Figure 2B show a TEM of the epithelium iayer of the amniotic membrane having a single layer of amniotic stem cells. The TEM is at 8200 X magnification.

[0036] Figure 3A is a scanning electron micrograph (SEM) of an amniotic membrane having amniotic stem celts.

[0 37| Figure 3B is a SEM of cryo-fractured amniotic membrane particles. £0038] Figure 4 is a scanning electron micrograph (SE ) of an amniotic membrane having pores between the amniotic membrane tissue.

[0039] Figure 5A is a representation of an exemplary tensi!ized amniotic membrane,

[0040] Figure 5B is a representation of two exemplary tensi!ized amniotic membranes being layered together.

[0044] Figure 6 shows a diagram of an exemplary method to apply a therapeutic composition, as described herein.

[0042] Figure 7 shows a diagram of a process to produce a fluid component comprising micronszed amniotic membrane particles.

[0043] Figure 8 shows a diagram of a process to produce a fluid component comprising a concentrated stem cell fluid.

[0044] Figure 9 shows a cross-sectional representation of an exemplary amniotic membrane configured over a treatment location,

[0045] Figure 10 shows a cross-sectional representation of an exemplar therapeutic composition comprising an amniotic membrane and fluid component configured over a treatment location.

[0046] Figure 11 shows a cross-sectional representation of an exemplary therapeutic composiie configured: over a treatment iocation wherein the iherapeutic composition comprises an amniotic membrane matrix component imbibed with a fluid component and a cover layer configured there over,

[0047] Figure 12 shows a cross-sectional representation of an exemplar therapeutic composite configured over a treatment Iocation wherein the therapeutic composite comprises a first matrix layer of amniotic membrane, a second matrix layer of a fluid component reservoir, and a third matrix layer thai is a cover layer.

[0048] Figure 13 shows a cross-sectional representation of an exemplary therapeutic composiie configured; over a treatment iocation wherein the iherapeutic composite comprises a first matrix Iayer of amniotic membrane imbibed with fluid component and a second matrix layer that is a support layer comprising

bioresorbable material.

[0049] Figure 14 shows a cross-sectional representation of an exemplary therapeutic composiie configured over a treatment location wherein the therapeutic composite comprises a first matrix layer of amniotic membrane imbibed with fluid component, a second matrix Saver that is a support !ayer and a third matrix iayer that comprises amniotic membrane.

[0050] Figure 15 shows an exemplary matrix component of a therapeutic composite configured around an artery and a fluid component being injected therein.

[0051] Figure 16 shows a diagram of the anatomy and various organs within the body.

[0052] Figure 17 shows a diagram of the circulatory system.

[0053] Figure 18 shows an exemplary fluid component being drawn from an enclosure by a syringe.

[0054] Figure 19 shows an exemplary catheter inserted through the femoral artery with the proximal end located at the heart.

[0055] Figure 20 shows flow cytometry analysis data for amniotic fluid as received and amniotic stem ceil concentrated fluid.

[0056] Corresponding reference characters indicate corresponding parts throughout the several views of the figures. The figures represent an illustration of some of the embodiments of the present invention and are not to be construed as limiting the scope of the invention in any manner. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0057] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Also, use of "a" or "an" are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[0058] Certain exemplary embodiments of the present invention are described herein and illustrated in the accompanying figures. The embodiments described are only for purposes of illustrating the present invention and should not be interpreted as limiting the scope of the invention. Other embodiments of the invention, and certain modifications, combinations and improvements of the described

embodiments, will occur to those skilled in the art and aii such alternate

embodiments, combinations, modifications, improvements are within the scope of the present invention.

[0059] As shown if FIG, 1 A the amniotic membrane surround a fetus in utero. As shown in F!G. 1 B, the amniotic membrane comprises an amnion portion and a chorion portion. As described herein, the amnion portion may be separated from the chorion, tn an exemplary embodiment the epithelium, or inner most layer of the amniotic membrane, is removed and used to produce particles for the therapeutic composite, as described herein. The particles may consists essentially of the epithelium, consists essentially of the epithelium and base membrane, consist essentiaiiy of the epithelium, base membrane and compact layer, or consist

essentiaiiy of epithelium, base membrane, compact layer, and fibroblast layer,

[0060] As shown in F!Gs, 2A and 2B, the epithelium layer of the amniotic membrane 20 has a single layer of amniotic stem ceils 48, The tissue around the amniotic stem cells may protect and enhance the viability of these stem cells when the epithelium is cryo-fractured to produce particles for the therapeutic composition.

[0061] As shown in FIG. 3A_« an amniotic membrane 20 comprises a plurality of amniotic stem cells 48.

[0062] As shown in FIG, 3B, particles of cryo-fractured amniotic membrane particles 40 are on the order of 0.2 to Ο.δμΓη in size. The average particle size shown is less than 2pm. There are no particles shown that are larger than 2pm and substantially all of the particles are less than 1 pm in size. The SEM shows that the micronized amniotic membrane particles are irregularly shaped. As shown, some of the particles have a planar surface.

[0063] As shown in FIG. 4 an amniotic membrane 20 comprises pores 29 between the amniotic membrane tissue. This porosity may be imbibed with a fluid component. In addition, an amniotic membrane may be stretched in one or more direction to tensilize the tissue, A tenstlize amniotic membrane may have a higher matrix tensile strength than an original un-tensilized amniotic membrane. In addition, a plurality of layers of amniotic membrane may be utilized to build strength in one or more directions. [0064] As shown in FIG. 5A, an amniotic membrane 20 has been stretched in one direction to form an elongated and more aligned amniotic tissue orientation. As shown in FIG, 5A, oriented tissue 23 is aligned horizontally and connecting tissue interconnects the oriented tissue. A tensilized amniotic membrane 21 may be stronger by unit weight in the oriented direction and may have a much higher elongation to break in the cross-oriented direction than a precursor amniotic membrane, before tensiSizing. The tensilized amniotic membrane 21 may be stretched as much as 120%, 150%, 175%, 200% of the original membrane length. The amniotic membrane may neck or narrow in the opposing direction of stretch. A stretched or tensilized amniotic membrane may be stretched over a long period of time to minimize tissue fracture. For example, an amniotic membrane may have a low load applied and may be stretched over a period of 10 minutes or more. 30 minutes or more, 1 hour or more, 8 hours or more, 1 day or more, 2 days more and any range between and including the durations provided. In addition, an amniotic membrane may be stretched while being hydrafed and or submerged In amniotic fluid or a plasticizing fluid. An amniotic membrane may be cross-linked after being stretched. The toad applied to tensi!ize an amniotic membrane may be a portion of the maximum tensile load required to fracture the amniotic membrane at a rate of l Omm/secood for a 2.54cm by 5.2cm sample having a 5cm gap. For example, a tensiiizing load applied may be no more than about 80%, no more than about 80%, no more tha about 50%, no more than about 25% of the maximum tensile load.

[0065] As shown in FIG. SB, a first tensilized amniotic membrane 20 is configured at a 90 degree offset from a second amniotic membrane 20'. This orientation of layering may provide for a much stronger therapeutic composite. In an alternative embodimeni, a plurality of layers of tensilized amniotic membrane may be aligned with the oriented tissue of a first layer being aligned with the oriented tissue of a second layer. A matrix component or a therapeutic composite, as described herein, may consist essentially of tensilized amniotic membrane.

[0066] Figure 6 shows a diagram of an exemplary method to apply a

therapeutic composite as described herein. As described herein, a fluid component ma be configured with a matrix component or may be applied after application of the matrix component to a treatment location.

[006?] As shown in FIG. 7, a process to produce a therapeutic composition, as described herein, comprises the steps of cryo-fractunng amniotic membrane fragments. As described, the amniotic membrane fragments may be cryo-fractured with a blunt object, such as a bar, that reduces shear and damage to the particles, in a preferred embodiment, the fragments are cryo-fractured with an object having substantially no sharp edges. The micronized particles are combined with any suitable carrier fluid to produce a therapeutic composite, in an exemplary embodiment, the micronized particles are dispersed in a fluid comprising stem ceil f!usd and amniotic stem cells. In another embodiment, the micronized particles are dispersed in a concentrated amniotic stem cell fluid.

[0068] As shown in FIG, 8, a process to produce a therapeutic composition, as described herein, comprises the steps concentrating amniotic stem cells in an amniotic fluid. An amniotic fluid may be processed in any suitable way to

concentrate the amniotic stem cells in the fluid. In an exemplary embodiment, as described in FIG. 5, the amniotic fluid is centrifuged to remove debris and excess liquid and concentrate the amniotic stem cells in the therapeutic composition,

[0069] As shown in FIG. 9, an exemplary therapeutic composition 11 is a therapeutic composite 10. The therapeutic composite 10 comprises an amniotic membrane 20, as a matrix component 2,configored over a treatment location 8, The matrix components in this embodiment consists essentially of amniotic membrane 20 and a fluid component 14 is coated onto the treatment surface 50 of the therapeutic composite. The fluid component 14 is not present on the outer surface 52 of the therapeutic composite 10.

[0070] As shown in FIG.10, an exemplary therapeutic composite 10 comprises an amniotic membrane 20 and a fluid component 14 imbibed therein, configured over a treatment location 18. The fluid component 14 comprises micronized amniotic membrane particles 40 and amniotic fluid 43, However, any suitable fluid carrier may be used to disperse the micronized amniotic membrane particles and or amniotic stem cells 46.

[0071 ] As shown in FIG. 11 , an exemplary therapeutic composite 10 is configured over a treatment location 18 wherein the therapeutic composite comprises an amniotic membrane 20 imbibed with a fluid component 14 and a cover layer 24 is configured there over. The matrix component 12 comprises a first matrix layer 30 and a second matrix layer 32. The second matrix layer is configured over said first matrix layer and comprises an overhang portion 38 that extends outside of the first matrix layer. The second matrix layer is attached to the tissue 19 by a attachment component 38, such as a staple, glue and/or sutures, for example. A matrix component or a layer of a matrix component may be configured to extend beyond a treatment location, whereby an outer area of the matrix component can be affixed to tissue, A cover layer may fully cover a first or under layer of matrix component or may only cover a portion of an layer thereunder, A cover layer may be a net or mesh or strands that extend across and over an under-layer, for example. An exemplary cover layer comprises pores or apertures 28 that allow fluid transfer to and from the treaiment location. Apertures may be small slits, holes, in an otherwise solid and impermeable matrix component or layer, or they may be pores in porous matrix component or layer. For example, an expanded polytetrafjuoroethySene membrane may have a mean flow pore size as measure by a Coulter Porometer (PM\ Industries), of less than 60um, less than 40um, less than 10um, less than 1 um and any range between and including the pore sizes provided. In one embodiment, the pores are sized to allow fluid to flow but retain cells, such as stem cells within the matrix component.

[0072] As shown in FIG. 12, an exemplary therapeutic composite 10 is configured over a treatment location 18 wherein the therapeutic composite comprises a matrix component 12 comprising a first matrix layer 12 of amniotic membrane 20, a second matrix layer 32 of a fluid reservoir layer 25, and a third matrix layer 34 that is a cover layer 24. The fluid reservoir layer comprises a matrix having porosity containing a fluid component 14 as described herein. As shown, a first fluid component 14 is configured within the first matrix layer 30. It is be noted that different compositions of a first and second fluid component may be configured in a matrix component 12. A first fluid component may comprise an amniotic stem cell concentrated fluid and a second fluid component may comprise micronized amniotic membrane dispersed in a fluid, for example. A reservoir layer may comprise a fluid component having stem cells, and these stem cells may be drawn from the reservoir layer as they are needed.

[0073] As shown in FIG. 13, a therapeutic composite 10 is configured over a treatment location 18 wherein the matrix component 12 comprises a first matrix layer

30 of amniotic membrane 20 imbibed with fluid component 14 and a second matrix layer 32 that is a support layer 22 comprising bioresorbable material 28. The support layer may be substantially impermeable to the fluid component configured in the first matrix component that is proximate a treatment location. In addition, an outer

I S surface 52 of a matrix component 12, or the surface facing away a treatment location, may be hydrophobic to reduce fluid ingress into the therapeutic composite. Bodil fluid ingress into a therapeutic composite ma dilute a fluid component comprises therein.

[0074] As shown in FIG. 14, an exemplary therapeutic composite 10 is configured over a treatment location 18 wherein the matrix component 12 comprises a first matrix layer 30 of amniotic membrane 20 imbibed with fluid component 14, a second matrix layer 32 that is a support layer 22 and a third matrix layer 34 that comprises amniotic membrane 20, A support layer is configured between amniotic membranes in this embodiment. As described herein, a matrix component may be provided with multiple layers attached and ready for orientation on a treatment location, or a plurality of matrix components may be applied, one after another, during the treatment procedure.

[0075] Any number of combinations of matrix components layers have been envisioned and are within the scope of the present invention. In addition, any number of different fiuid components may be incorporated into a therapeutic composite as described herein.

[0076] As shown in FIG. 15, an exemplary therapeutic composite 10 is configured around an constrictable body part 64, such as artery, intestine ureter and the like, and a fluid component 14 is being injected therein. This type of procedure may reduce and/or eliminate aneurisms, A matri component may be a sheet of materia] having a substantiall pianar top and bottom surface and substantially uniform thickness therebetween. A sheet of matrix composite may be supple and ma be configured around a cylindrical treatment location, such as an artery or vein, in another embodiment, a matrix component sheet is applied externally over a treatment location, such as to the epicardium. Any constrictable body part, or body part that can be wrapped with a matrix component, may be treated by wrapping a matrix component around the constrictabie body part and alternatively injecting the body part with a therapeutic fluid, as described herein,

[0077] Figure 8 shows a diagram of the anatomy and various organs within the body that may be treated with a therapeutic composite as described herein. A therapeutic composite, as described herein, may be introduced into any anatomy shown in FIG. 18 b open surgery, topical application, or transcatheter. A deliver vehicle such as a stent or balloon may be used with a therapeutic composite, as described herein. For example, a therapeutic composite may be introduced into any portion of the urinary or digestive system, including the bladder, ureter, urethra, small intestine, large intestine, stomach, esophagus, mouth, tongue, colon, rectum, and the like.

[0078] Figure 17 shows a diagram of the circulatory system where a therapeutic composite may be introduced into the body through transcatheter.

[0079] Figure 18 shows an exemplary fluid component 14 being drawn from an enclosure 70 by a syringe 80. The fluid component comprises micronized particles 40 of amniotic membrane 20 and stromal vascular fraction 48 in a concentrated amniotic stem cell fluid 44. The needle may be any suitabl size, however in a preferred embodiment the needle is no larger than a 20 gauge needle,

[0080] As shown in FIG, 19, a catheter is inserted into the femoral artery and the proximal end of the catheter is located at the heart. A therapeutic composite may be introduced through a catheter to a treatment location within the body, including any suitable organ as shown in FIG. 16, A catheter may be configured with an injection implement at the proximal end to enable the therapeutic composite to be injected into tissue, such as tissue of an organ.

[0081] Figure 20 shows flow cytometry analysis data for amniotic fluid as received and amniotic stem celt concentrated fluid as described herein. Flow cytometry was performed on four different liquid samples from different donors. The analyses shows that the expression level of mesenchymal stem ceil surface antigens is consistent between donors with CD44 being positive and CD73 being strongly positive while CO90 and CD105 are low positive. The level of expression is maintained between the processed samples concentrated sample 1 and

concentrated sample 2 and unprocessed samples (Fresh Amniotic Fluid 1&2), suggesting no cell loss during the manufacturing process and preservation of potency. What is also interesting is that CD73 is expressed the most. It has been reported that mesenchymal stem cell migration is controlled by CD73 and therefore It is speculated that a high !evei of CD73 expression promotes ceil migration and the ability of the cells to home to tissue sites of repair or to participate in healing responses.

Example 1 £0082] Three fluid components were made and cell viability was measured as reported in Table 1. Three amniotic membrane samples, obtained from three separate donors, were cryo-fractured and dispersed in fluid to create a fluid components, as described herein.

[0083] A fluid component of the therapeutic composite was prepared by concentrations amniotic stem ceils in a ceil suspension solution. A 1mi sample of an unprocessed amniotic fluid was used to measure initial ce!i count and viability. The amniotic fluid was then separated into 50m! steriie centrifuge tubes and centrifuged two times at 400xg for 10 minutes at ambient temperature. Cell pellet from each tube was washed with 20m! of Plasma Lyte-A, from Baxter Inc., between

centrifugation. Supernatant was removed and cells were re-suspended In a predetermined volume of cell suspension solution. Plasma Lyte-A, to obtain a final product cell concentration of 1 x 10^s cell/mi.

[0084] Cryo-fractured particles of amniotic membrane were prepared for dispersion in the fluid component. Three amniotic membranes were obtained and rinsed using Plasma Lyte~A and transferred to a cutting board. Using blunt dissection, chorion was removed from the amniotic membrane and any remaining debris/blood was removed using sterile laps. The amniotic membrane dimensions were measured using a sterile stainless steel ruler. The amount of amniotic membrane needed to obtain a concentration of 1cm²/m( of therapeutic solution was retained and placed on a sterile drying rack and allowed to dry for one hour. The amniotic membrane was then cut into smalt pieces, less than a 1cm² and placed inside a milling chamber containing a blunt impactor. The cryo-miSI used was from SPEX Sample Prep Inc., 697QEFfv! Enclosed Freezer/Sv i yodel 8970D.

[0085] The milling chambers were placed Inside the cryomill and the amniotic membrane was micronized. The frequency of the impactor was 8 cycles per second, the preceding time was five minutes, the grinding time was three minutes and the intermediate cooling time was two minutes, After the micronization of the amniotic membrane was complete, the chambers were removed from the cryomill and allowed to warm at room temperature for one hour. The cryo-fractured amniotic membrane was then dispersed in 100ml of fluid component prepared as described in this example. The final therapeutic composite was prepared by combining 100ml or the fluid component and micronized amniotic membrane with equal volume (100ml), of cryprotectant solution, CryoStor 10, available from Sigma-Aldrich, Using a repealer pipet, cryovia!s were then filled at the desired volume. The therapeutic solution was maintained at 4°C during the via! filling process to preserve ceii viability.

[0086] The cryoviais were then cryop reserved using a controlled rate freezer. The controlled freezing protocol; cool at a rate of 1.C C/min until chamber reached - 4°C, cool at rate of 25.0°C/min until chamber reached -40°C, warm at a rate of 1 G,Q°C/min until chamber reached -12^aC, cool at rate of 1 ,0°C/min until chamber reached -40°C, and cool at rate of 10.0°C/min until chamber reached ~90^i:iC.

Cryoviais were then placed into cryo-boxes and transferred to a -80.0°C freezer.

[0087] Thawing of the cryoviais was performed and cell viability was again measured. Cell viability pre and post cryopreservation is reported in Table 1. The cryoviais were removed from the ~S0,0°C freezer and allowed to thaw at room temperature until the fluid components in the vial had a slushy consistency, or approximately three minutes for a 1 ml sample. An equal amount of cold Plasma Lyte-A was added to the sample for a 1 ;2 dilution. Samples were mixed and a small aliquot was used to perform cell count and viability enumeration. Cell count and viability was assessed using Trypan Blue.

Definitions:

[0088] Micro ized particles, such as micronized amniotic membrane particles, as used herein, means that the particles have an average particle size of less than 1 pm_: and in some cases have an average pa ticie size of less than Ο.δμηι. Particle size may be measured by analysis of scanning electron micrographs

[0089] It will be apparent to those skilled in the art that various modifications, combinations and variations can be made in the present invention without departing from the spirit or scope of the invention. Specific embodiments, features and elements described herein may be modified, and/or combined in any suitable manner. Thus, it is intended that the present invention cover the modifications, combinations and variations of this invention provided they come within the scope of the appended claims and their equivalents.

?9

Claims

What is claimed is;

1. A therapeutic composition comprising a fluid component comprising amniotic fluid.

2. The therapeutic composition of claims 1 , wherein the fluid component

comprises a concentration of amniotic stem ceils of at least 0.1 x 10⁶ per milliliter of said fluid component.

3- The therapeutic composition of claims 1 , wherein the fluid component

comprises a concentration of amniotic stem ceils of at least 0.5 x 10⁶ per milliliter of said fluid component,

4. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stern cells of at least 1 x 10⁶ per milliliter of said fluid component.

5. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stem ceils of at least 5 x 10^s per milliliter of said fluid component,

6. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stem cells of at least 10 x 10^fi per milliliter of said fluid component.

7. The therapeutic composition of claims , wherein the fluid component

comprises an aceilular fiuid component.

8. The therapeutic composition of claims 1 , wherein the fluid component

comprises an aceilular amniotic fluid,

9. The therapeutic composition of claims 1 , wherein the fluid component consists of an aceilular amniotic fluid.

10. The therapeutic composition of claims 1 , wherein the fluid component has a viscosity of no more than about 50 Pa sec.

11.The therapeutic composition of claims 1 , wherein said fluid component is a paste having a viscosity of more than about 50 Pa sec.

12, The therapeutic composition of claims 1 , wherein the f!uid component

comprises a plurality of protein markers including CD44, CD105, CD73, and CD90 proteins.

13. The therapeutic composition of claims 1 , wherein the fluid component

comprises: a. growth factors; and

b. proteins.

, The therapeutic composition of claims 1 to 13, further comprising micronized amniotic membrane particles,

, he therapeutic composition of claims 14, wherein the particles consist essentially of micronized amniotic membrane particles.

, The therapeutic composition of claim 14. wherein the micronized amniotic membrane particles consist essentially of amnion and are essentially free of chorion,

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium .

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium and amnion basement membrane.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium, amnion basement membrane and compact layer.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium, amnion basement membrane, compact layer and fibroblast layer.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are hydrated and have a percent hydration of a least 20 percent.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are dece!Sularized particles.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 250pm.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 150pm.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are irregularly shaped. 28, The therapeutic composition of daim 14, wherein the micronized amniotic membrane particles are planar in shape, having a first pianar surface and a second planar surface,

27. The therapeutic composition of ciaim 14, having a concentration of micronized amniotic membrane particles of at least about 1.Omg/mi of therapeutic composition,

28. The therapeutic composition of daim 14, having a concentration of micronized amniotic membrane particles of at least about 5.Qmg/mi of therapeutic composition.

29. The therapeutic composition of claim 14, having a concentration of micronized amniotic membrane particles of at least about IG.Qmg/ml of therapeutic composition,

30. The therapeutic composition of ciaim 14, wherein the micronized amniotic membrane particles comprise:

a. collagen; and

b, growth factors.

31. A therapeutic composition of claims 1 to 30 comprising;

a. a matrix component comprising amniotic membrane;

32. The therapeuiic composition of claim 31 , wherein the amniotic membrane consists essentially of amnion and is essentially free of chorion.

33. The therapeutic composition of ciaim 31 , wherein the amniotic membrane consists essentially of epithelium,

34. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentially of epithelium and amnion basement membrane,

35. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentially of epithelium, amnion basement membrane and compact iayer,

36. The therapeutic composition of ciaim 31 , wherein the amniotic membrane consists essentially of epithelium, amnion basement membrane, compact Iayer and fibroblast layer.

37. The therapeutic composiiion of claim 31 , wherein the amniotic membrane Is hydrated and has a percent hydration of at least 20 percent. 38, The therapeutic composition of ciaim 31, wherein the amniotic membrane are is a deceiluiarized amniotic membrane.

39, The therapeutic composition of ciaim 31 , wherein the amniotic membrane is tensiiized in one direction to produce oriented amniotic tissue,

40, The therapeutic composition of ciaim 31 _» wherein the amniotic membrane is tensiiized bi-axiaiiy.

41.The therapeutic composition of ciaim 31 to 40, comprising a first layer of amniotic membrane and a second matrix component layer.

42. The therapeutic composition of ciaim 41 , wherein the second matrix

component layer is configured for placement over said first layer of amniotic membrane,

43. The therapeutic composition of ciaim 41 , wherein the second matrix

component layer is an amniotic membrane.

44. The therapeutic composition of ciaim 41 , wherein the second matrix

component layer is a bioresorbable materia!.

45. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a non-bioresorbable polymer materia!.

46. The therapeutic composition of claim 41 , wherein the second matrix

component layer is substantially non-permeable to a fiuid component retained in the first layer of amniotic membrane.

47. The therapeutic composition of ciaim 41 , wherein the second matrix

component layer is a fluid reservoir layer, whereby said second matrix component layer has porosity that is at least 50% filled with a fluid

component.

48. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a support layer, having a tensile break strength of at least two times that of a first layer of amniotic membrane tensile break strength,

49. The therapeutic composition of ciaim 41 , wherein the first layer of amniotic membrane is a tensiiized amniotic membrane and a second matrix

component layer a tensiiized amniotic membrane.

50. The therapeutic composition of claims 1 to 49, further comprising a plurality of progenitor cells.

51.The therapeutic composition of daims 1 to 49, further comprising a plurality of vascular fraction cells.

52, The therapeutic composition of claims 1 to 49, further comprising a plurality of progenitor ceils derived from vascular fraction eel is.

53, The therapeutic composition of daims 1 to 49, further comprising

preadipocytes, mesenchymal stem ceils and endothelial progenitor cells from a stromal vascular fraction.

54, The therapeutic composition of claims 1 to 49, further comprising

mesenchymal stem cells,

55, The therapeutic composition of claims 1 to 49, further comprising bone

marrow aspirate,

56, The therapeutic composition of claims 1 to 49, further comprising platelet rich plasma.

57, The therapeutic composition of claim claims 1 to 49, further comprising an oxygen-carrier component.

58, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises a perfluorocarbon.

59, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises a peilluoroperhydrophenanthrene.

60, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises is bonded to a matrix component.

61 , The therapeutic composition of claims 57, wherein the oxygen-carrier is a perfluorocarbon and wherein the fluid component is an emulsion,

62, The therapeutic composition of claim 1 to 81 _« wherein the therapeutic

composition is a thawed therapeutic composition from a cryopreserved state,

83. A method of treating diabetes by introducing any suitable therapeutic

composition as described in any of claims 1 to 61 to a pancreas.

64, A method of treating kidney failure by introducing any suitable therapeutic composition as described in any of ciaims 1 to 61 to a kidney.

65. A method of treating liver failure by introducing any suitable therapeutic

composition as described in any of claims 1 to 61 to a kidney.

68. A method of treating renal failure by introducing any suitable therapeutic composition as described in any of claims 1 to 81 to a kidney.

67. The method of claims 83 to 88, wherein the therapeutic composition is

introduced intravenously.

88, The methods of claims 63 to 66, wherein the therapeutic composition is

introduced by a topical application.

69. A method of inducing immunomoduiaiion to a Sung by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a lung.

70. A method of inducing an anti-inflammatory response of a lung by introducing any suitable therapeutic composition as described in an of claims 1 to 62 to a lung.

71. A method of treating COPP by providing a introducing any suitable

therapeutic composition as described in any of claims 1 to 62 to a lung.

72. A method of treating lung fibrosis by introducing any suitable therapeutic

composition as described in any of claims 1 to 62 to a Sung.

73. A method of ireating acute tung injury by introducing any suitable therapeutic composition as described in any of claims 1 to 62 intravenously.

74. A method of treating a transplanted lung by introducing any suitable

therapeutic composition as described in any of claims 1 to 82 to a lung.

75. A method of treating pulmonary hypertension by Introducing any suitable

therapeutic composition as described in any of claims 1 to 62 to a lung.

76. A method of treating ventilator induced !ung injury by introducing a therapeutic composition as described in any of claims 1 to 62 to a lung.

77. A method of ireating acute respiratory disease syndrome (A DS) by

introducing any suitable therapeutic composition as described in an of c!aims 1 to 62 to a Sung.

78. A method of treating bronchitis by introducing any suitable therapeutic

composition as described in any of claims 1 to 62 to a lung.

79. A method of treating alveolitis by introducing any suitable therapeutic

composition as described in any of claims 1 to 62 to a lung.

80. A method of ireating chronic parenchymal lung disease by introducing any suitable therapeutic composite as described in any of claims 1 to 62 to a lung.

81. A method of treating pleura! lung disease by introducing any suitable therapeutic composition as described in any of claims 1 to 82 to a lung.

82. A method of treating trans-bronchial parenchymai disease by introducing any suitable therapeutic composition as described in any of daims 1 to 62 to a Sung.

83. A method of treating post-operative lung recovery and scaring by introducing any suitable therapeutic compositio as described in any of claims 1 to 62 to a lung.

84. A method of treating empyema by introducing any suitable therapeutic

composition as described in any of claims 1 to 62 to a lung.

85. The method of daims 69 to 84, wherein the therapeutic composition is

introduced intravenously to said lung.

86. A method of claims 69 to 84_; wherein the therapeutic composition is

introduced into a pulmonary artery.

87. A method of claims 69 to 84, wherein the therapeutic composition is

introduced through a central venous catheter.

88. A method of claims 69 to 84, wherein the therapeutic composition is

introduced through a trans-bronchial catheter,

89. A method of claims 69 to 84, wherein the therapeutic composition is

introduced though a peripheral venous catheter.

90. A method of claims 69 to 84, wherein the therapeutic composition is

introduced by a topical application,

91. A method of treating the brain by introducing any suitable therapeutic

composition as described in any of claims 1 to 62 to a portion of the brain,

92. A method of treating the brain of claims 91 wherein the therapeutic

composition further comprises mannito!.

93. A method of treating an ischemic stroke by introducing any suitable

therapeutic composition as described in any of claims 1 to 62 to a brain.

94. A method of treating a traumatic brain injury a by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a brain.

95. A method of treating a post-operative brain recovery and scaring by introducing any suitable therapeutic composition as descnbed in any of claims 1 to 82 to a brain.

96. A method of treating a Parkinsons by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a brain,

97. A method of treating a neuro degenerative disease by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a brain.

98. A method of treating a chronic traumatic encephalopathy by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a brain,

99. A method of claims 91 to 98, wherein the therapeutic composition is

introduced by a topical application to a portion of a brain.

100. A method of claims 91 to 98, wherein the therapeutic composition is introduced by topical application and a subsequent injection of a fluid component.

101. A method of claims 91 to 98, wherein the therapeutic composition is introduced by intravenously.

102. A method of claims 91 to 98, wherein the therapeutic composition is introduced through a shunt.

103. A method of treating strictures in the ureter by placement of any

suitable therapeutic composition as described in any of claims 1 to 62 to said ureter,

104. A method of treating strictures in the urethra by placement of any

suitable therapeutic composition as described in any of claims 1 to 62 to said urethra.

105. A method of treating erectile dysfunction by introducing any suitable therapeutic composition as described in any of claims 1 to 62 the base of the penis.

106. A method of treating Peyronie's disease by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a portion of the penis.

107. A method of treating Peyronie's disease by introducing any suitable therapeutic composition as described in any of claims 1 to 82 through injection into a portion of the penis,

108. A method of preventing adhesions of an organ after an incision by the application of a therapeuiie composite comprising an amniotic membrane over the incision,

109. A method of treating a constrictabie organ, by wrapping a therapeuiie composition comprising an amniotic membrane around the constrictabie organ.