Compositions and methods relating to mixed fetal support tissue

Abstract

Described herein is a composition, method comprising a mixed fetal support tissue, wherein the mixed fetal support tissue comprises a therapeutically effective amount of a native HC-HA / PTX3 complex.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Application No. 63 / 334,283, filed on April 25, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Provided herein are compositions and methods related to mixed fetal support tissues. Because fetal support tissues have shown great potential to combat a variety of clinical indications, there has been a great interest in the development of fetal support tissue products. However, the clinical and commercial potential of fetal support tissue products depends on the effective processing of tissue products and the uniformity and efficacy of the final product. When processing fetal support tissues, the preservation of bioactive components is critical. For example, differences in processing methods can change the structure and biochemical composition of fetal support tissues and can weaken the activity of key signaling molecules that are critical to the intended therapeutic activity. Disclosed herein are compositions and methods for separating, processing and generating fetal support tissue products and / or preparations that maintain the efficacy and stability of tissue bioactive factors.

[0004] Typically, conventional protocols do not utilize the mixing of cells, tissues or biological products from more than one human donor because there is an infectious disease risk and it is difficult to establish manufacturing and processing / production facilities that meet regulatory standards. The present disclosure advantageously describes the mixing of donor fetal support tissues. First, the practice of mixing biological products from multiple donors reduces the inherent variability between different donors. The mixing of donor tissues controls the effectiveness of each individual donor, improves consistency and promotes the efficiency of further qualitative, sampling and clinical testing. In addition, the mixing of donor samples enriches the fetal support tissue components. A method for generating mixed fetal support tissues from multiple donors is disclosed herein. In some embodiments, the freeze drying of fetal support tissue significantly reduces the degradation of tissue bioactive components in fetal support tissue and concentrates bioactive tissue components, such as, for example, increasing the stability and concentration of bioactive components contained in fetal support tissue materials, including HA complexes (HC-HA / PTX3), cytokines, growth factors and any other bioactive factors found in fetal support tissues. Summary of the invention

[0005] In one aspect, a composition comprising a mixed fetal support tissue is described herein, wherein the mixed fetal support tissue comprises a therapeutically effective amount of a natural HC-HA / PTX3 complex. In some embodiments, the composition comprises a mixed fetal support tissue, wherein the fetal support tissue comprises a therapeutically effective amount of a natural HC-HA / PTX3 complex. In some embodiments, wherein the mixed fetal support tissue improves the uniformity of the natural HC-HA / PTX3 complex in the composition. In some embodiments, the mixed fetal support tissue comprises fetal support tissue from multiple donors. In some embodiments, a composition is disclosed herein, wherein the mixed fetal support tissue comprises fetal support tissue from at least three donors. In some embodiments, the mixed fetal support tissue comprises fetal support tissue from at least five donors. In some embodiments, the mixed fetal support tissue comprises fetal support tissue from at least ten donors. In some embodiments, the composition exhibits a therapeutic efficacy determined by one or more assays including HA content assay, BCA total protein assay, PTX3 content assay, ODI-TRAP assay, M2 polarization assay, or WST-1 assay.

[0006] In some embodiments, the therapeutic efficacy is determined by an ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 200 μg / ml, 250 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1,000 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to a positive control in an ODI-TRAP assay.

[0007] In some embodiments, the positive control is RANKL treatment for ODI-TRAP. In some embodiments, the efficacy of the treatment is determined by an M2 assay. In some embodiments, the efficacy of the treatment is determined by an NO assay.

[0008] In some embodiments, therapeutic efficacy is determined by various assays, such as, but not limited to, the WST-1 assay.

[0009] In some embodiments, the therapeutic efficacy is determined by BCA protein assay. In some embodiments, the therapeutic efficacy is determined by HA content assay. In some embodiments, the HA content of the composition is at least 75 μg / ml. In some embodiments, the HA content of the composition is at least 90 μg / ml. In some embodiments, the total protein content of the composition is at least 250 μg / ml. In some embodiments, the mixed fetal support tissue is freeze-dried. In some embodiments, the mixed fetal support tissue is freeze-dried before the mixed fetal support tissue. In some embodiments, the mixed fetal support tissue is freeze-dried after the mixed fetal support tissue. In some embodiments, the mixed fetal support tissue composition has been frozen or cryopreserved. In some embodiments, the mixed fetal support tissue includes a particle size of about 0.01 microns (μm) to about 240 μm in diameter. In some embodiments, the mixed fetal support tissue includes an average particle size of about 0.5 μm. In some embodiments, the mixed fetal support tissue includes placenta, umbilical cord, placental amnion, umbilical cord amnion, Wharton's jelly, chorion or amnion-chorion, or any combination thereof. In some embodiments, the mixed fetal support tissue includes placenta amnion, umbilical cord or both. In some embodiments, the mixed fetal support tissue is crushed before mixing. In some embodiments, the mixed fetal support tissue is crushed after mixing. In some embodiments, the mixed fetal support tissue is fragmented. In some embodiments, the mixed composition is filtered or ultrafiltered. In some embodiments, the mixed fetal support tissue is decellularized. In some embodiments, the mixed fetal support tissue is inactivated. In some embodiments, the mixed fetal support tissue is a gel extract. In some embodiments, the mixed fetal support tissue is powdered. In some embodiments, the mixed fetal support tissue contains 5% or more water by weight. In some embodiments, the mixed fetal support tissue contains up to 15% water by weight. In some embodiments, the mixed fetal support tissue is obtained from frozen or once frozen fetal support tissue. In some embodiments, the mixed fetal support tissue is substantially free of veins or arteries. In some embodiments, all cells of the mixed fetal support tissue are substantially dead. In some embodiments, the mixed fetal support tissue is terminally sterilized, gamma irradiated, filtered, electron beam sterilized, or a combination thereof. In some embodiments, the composition is provided as a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is normal saline.

[0010] The present application also discloses a method of treating a degenerative disease in an individual, comprising administering to the individual the mixed fetal support tissue described herein.

[0011] Described herein is a method of treating inflammation in an individual, comprising administering to the individual a mixed fetal support tissue as described herein. In another aspect, disclosed herein is a method of promoting wound healing in an individual, comprising administering to the individual a mixed fetal support tissue as described herein.

[0012] In addition, a method for treating osteoarthritis in an individual using a composition comprising mixed fetal support tissue is disclosed herein on the other hand, the method comprising administering mixed fetal support tissue to the individual. In some embodiments disclosed herein, the osteoarthritis may be osteoarthritis of the knee joint. In some embodiments, the osteoarthritis may be osteoarthritis of the facet joint. In some embodiments, the osteoarthritis may be osteoarthritis of the ankle joint. In some embodiments, the osteoarthritis may be osteoarthritis of the hip joint. In some embodiments, the osteoarthritis may be osteoarthritis of the shoulder joint. In some embodiments, the osteoarthritis may be osteoarthritis of the elbow joint. In some embodiments, the osteoarthritis may be osteoarthritis of the wrist joint. In some embodiments, the osteoarthritis may be osteoarthritis of the small joints of the hand. In some embodiments, the osteoarthritis may be osteoarthritis of the small joints of the foot. In some embodiments, the osteoarthritis may be osteoarthritis of the cervical spine. In some embodiments, a method for preparing a composition of mixed fetal support tissue is disclosed herein, comprising obtaining fetal support tissue from a plurality of donors and mixing the fetal support tissue from a plurality of donors. In some embodiments, the fetal support tissue from a plurality of donors is freeze-dried before mixing. In some embodiments, the fetal support tissue from a plurality of donors is freeze-dried after mixing. In some embodiments, the method of preparing a freeze-dried composition further comprises processing fetal support tissue from multiple donors by fragmentation, pulverization, micronization, or a combination thereof. In some embodiments, the method further comprises devitalizing fetal support tissue from multiple donors. In some embodiments, the method further comprises decellularizing fetal support tissue from multiple donors. In some embodiments, fetal support tissue from multiple donors is not dehydrated. In some embodiments, fetal support tissue from multiple donors is not dehydrated to contain less than 20% water by weight. In some embodiments, fetal support tissue from multiple donors is not dehydrated to contain less than 15% water by weight.

[0013] In certain embodiments, disclosed herein is a method for validating a mixed fetal support tissue product, the method comprising performing a validation assay on the mixed fetal support tissue, wherein the validation assay determines the amount and / or therapeutic efficacy of the native HC-HA / PTX3 complex. In some embodiments, the native HC-HA / PTX3 has therapeutic activity. In some embodiments, the validation assay comprises one or more of a HA content assay, a PTX3 content assay, an ODI-TRAP assay, a BCA protein assay, an M2 polarization assay, a NO assay, or a WST-1 assay. In some embodiments, the validation assay comprises an ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 200 μg / ml, 250 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1,000 μg / ml, the fetal support tissue product mixed in the ODI-TRAP assay shows statistically significant inhibition of TRAP activity compared to the positive control. In some embodiments, the positive control is RANKL treatment. In some embodiments, the therapeutic efficacy is determined by the M2 assay. In some embodiments, the therapeutic efficacy is determined by the NO assay. In some embodiments, the therapeutic efficacy is determined by the WST-1 assay. In some embodiments, the therapeutic efficacy is determined by the BCA protein assay. In some embodiments, the therapeutic efficacy is determined by the HA content assay. In some embodiments, the therapeutic efficacy is determined by the PTX3 content assay. In some embodiments, the HA content of the composition is at least 75 μg / ml. In some embodiments, the HA content of the composition is at least 90 μg / ml. In some embodiments, the total protein content of the composition is at least 250 μg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A-1B A flow chart is shown illustrating an example of a process flow or method for producing a fetal support tissue product. The process flow illustrates the steps of procuring a donor sample, cleaning and processing ( Figure 1A ) to primary packaging in powder or micronized form ( Figure 1B ) processing steps, as disclosed herein, by pooling multiple donors for processing to reduce sample variability, improve consistency, increase yield, improve efficacy, and uniform potency.

[0015] Figure 2A-2C A flow chart is shown illustrating an example of a process flow or method for producing a fetal support tissue product in liquid form (API processing) by blending multiple donors to increase yield and uniform potency. A process for sample reception and cleaning is shown ( Figure 2A ), sample handling and mixing process ( Figure 2B) and the receipt, storage and packaging of medicines ( Figure 2C ).

[0016] Figure 3 Examples of cell morphology analysis and ODI-TRAP assays of fetal support tissue products are shown.

[0017] Figure 4 Examples of cell morphology analysis and M2 IL-12 assays of fetal support tissue products are shown.

[0018] Figure 5 Examples of cell morphology analysis and an exemplary M2 NO assay for a fetal support tissue product are shown.

[0019] Figure 6A-6C Examples of linear analysis of the dose dependence of hyaluronic acid polysaccharide ("HA") concentration in three potency assays are shown: WST-1 assay ( Fig. 6A ); TRAP assay ( Figure 6B ); M2 IL-12 assay ( Figure 6C ). Abbreviations MS = MAU / saline; M / W = MAU / WFI; M / S dlyz = MAU / dialyzed saline; MAU ​​= fragmented amniotic membrane and umbilical cord; WFI = water for injection.

[0020] Figure 7 Examples of hyaluronic acid (HA) quantification (μg / ml HA) of samples obtained from individual or single donor batches and mixed batches are shown.

[0021] Figure 8A-8B An example of a Western blot analysis of a fetal support tissue product is shown. Samples obtained from single donor batches and pooled donor batches were evaluated in a Western blot assay to qualitatively assess the heavy chain 1 (HC1; Fig. 8A ) and pentraxin (PTX3; Figure 8B )Changes in polymers / macromolecules.

[0022] Fig. 9 An example of a qualitative analysis of HA is shown, evaluating molecular weight variations in samples from agarose gel electrophoresis from single donor batches (SD-1 to SD-4; non-mixed fetal support tissue in lanes 3-7) and mixed donor batches (P1-P3; mixed fetal support tissue (pFST) in lanes 8-10).

[0023] Figures 10A-10B Shown are the results for batches from single / individual donors ( Fig. 10A ) and mixed donor batches ( Fig. 10B) samples.

[0024] Fig.11 An example of bicinchoninic acid (BCA) quantification assay performed on samples from single / individual donor batches and pooled donor batches is shown. DETAILED DESCRIPTION

[0025] Specific Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0027] As used herein, in some embodiments, ranges and amounts are expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 μg" means "about 5 μg" as well as "5 μg". In general, the term "about" includes amounts that are expected to be within the range of experimental error.

[0028] As used herein, "fetal support tissue" refers to any isolated tissue derived from tissue used to support fetal development. Examples of fetal support tissue include, but are not limited to: (i) placental amniotic membrane (PAM) or substantially isolated PAM, (ii) umbilical cord amniotic membrane (UCAM) or substantially isolated UCAM, (iii) chorion or substantially isolated chorion, (iv) amnion-chorion or substantially isolated amnion-chorion, (v) placenta or substantially isolated placenta, (vi) umbilical cord or substantially isolated umbilical cord, or (vii) any combination thereof. In some embodiments, the fetal support tissue is selected from the group consisting of placental amniotic membrane (PAM), umbilical cord amniotic membrane (UCAM), chorion, amnion-chorion, placenta, umbilical cord, and any combination thereof. In some embodiments, the fetal support tissue includes the umbilical cord. The fetal support tissue includes any form of fetal support tissue, including fresh, cryopreserved, terminally sterilized, micronized, freeze-dried fetal support tissue, or powder produced by grinding the fetal support tissue. In some embodiments, the fetal support tissue is a ground, pulverized, fragmented, explant, slice, powder, gel, homogenate, extract, cryo-pulverized, decellularized, sieved, freeze-dried, dehydrated or liquefied, vaporized, micronized, or terminally sterilized product.

[0029] Fetal support tissues include the amniotic membrane and the umbilical cord, which contain a variety of innate biological factors that can be used for a variety of purposes, including reducing inflammation and scar formation to promote regenerative wound healing. The biological factors that can be found in fetal support tissues can contain components such as extracellular matrix, growth factors, and cytokines. Among the extracellular matrix, hyaluronic acid (HA) and HA-containing complexes, namely native HC-HA / PTX3, are uniquely and abundantly present in the amniotic membrane (AM) and the umbilical cord (UC). The HC-HA / PTX3 complex—a high molecular weight (HMW) hyaluronic acid polysaccharide (HA) covalently linked to the heavy chain (HC) 1 from inter-α-trypsin inhibitor and further complexed with pentraxin 3 (PTX3)—is a key active component of AM and UC, responsible for the above-mentioned role in promoting regenerative wound healing. Therefore, there is a need to produce fetal support tissue products with high yields of HC-HA / PTX3, HA, and other target proteins. There is also a need to produce fetal support tissue products using treatments that reduce or prevent degradation of the HC-HA / PTX3 complex and other target proteins. Preventing degradation of HC-HA / PTX3, HA, and other target proteins is important because degradation of such proteins would render the fetal support tissue product unsuitable for use.

[0030] As used herein, "powder" refers to a substance in the form of fine dry particles. In some embodiments, the size of the particles is not uniform. In some embodiments, the size of the particles is substantially uniform.

[0031] As used herein, "grinding" refers to any method of reducing fetal support tissue to small particles or powder. The term grinding includes pulverizing, homogenizing, rasping, milling, planing, pounding, and crushing.

[0032] As used herein, "placenta" refers to the organ that connects the developing fetus to the maternal uterine wall to allow absorption of nutrients, elimination of waste, and gas exchange through the maternal blood supply. The placenta consists of three layers. The innermost placental layer surrounding the fetus is called the amnion. The allantois is the middle layer of the placenta (derived from the embryonic hindgut); blood vessels derived from the umbilical cord pass through this membrane. The outermost layer of the placenta, the chorion, contacts the endometrium. The chorion and allantois fuse to form the chorioallantoic membrane.

[0033] As used herein, "chorion" refers to a membrane formed by the extraembryonic mesoderm and two layers of trophoblast. The chorion consists of two layers: an outer layer formed by the trophoblast and an inner layer formed by the somatic mesoderm; the amnion is in contact with the latter. The trophoblast consists of an inner layer of cubic or prismatic cells (i.e., cytotrophoblast or Langhans layer) and an outer layer of protoplasm rich in nuclei (without cell borders), i.e., syncytiotrophoblast. The avascular amnion adheres to the inner layer of the chorion.

[0034] As used herein, "amniotic-chorionic" refers to a product comprising amnion and chorion. In some embodiments, the amnion and chorion are not separated (i.e., the amnion is naturally adhered to the inner layer of the chorion). In some embodiments, the amnion is initially separated from the chorion and then combined with the chorion during processing.

[0035] As used herein, "umbilical cord" refers to the organ that connects the developing fetus to the placenta. The umbilical cord is composed of Wharton's Jelly, a jelly-like substance composed primarily of mucopolysaccharides. It contains a vein that carries oxygenated, nutrient-rich blood to the fetus and two arteries that carry deoxygenated, nutrient-poor blood away.

[0036] As used herein, "placental amniotic membrane" (PAM) refers to amniotic membrane derived from the placenta. In some embodiments, the PAM is substantially isolated.

[0037] As used herein, "umbilical cord amniotic membrane" (UCAM) refers to the amniotic membrane that has extended to become part of the umbilical cord. UCAM is a translucent membrane. UCAM has multiple layers: an epithelial layer, a basement membrane; a dense layer; a fibroblast layer; and a spongy layer. It lacks blood vessels or a direct blood supply. In some embodiments, UCAM includes Wharton's jelly. In some embodiments, UCAM includes blood vessels and / or arteries. In some embodiments, UCAM includes Wharton's jelly and blood vessels and / or arteries.

[0038] As used herein, "human tissue" refers to any tissue derived from the human body. In some embodiments, the human tissue is a fetal support tissue selected from the group consisting of placenta amnion, umbilical cord, umbilical cord amnion, chorion, amnion-chorion, placenta, or any combination thereof.

[0039] As used herein, "minimal manipulation" means (1) for structural tissues, processing that does not change the original relevant characteristics of the tissue that are relevant to the use of the tissue in reconstruction, repair, or replacement; and (2) for cells or non-structural tissues, processing that does not change the relevant biological characteristics of the cells or tissues.

[0040] The term "fresh fetal support tissue" refers to fetal support tissue that is less than 72 hours after birth and is in substantially the same form as after birth. In some embodiments, fresh fetal support tissue includes fetal support tissue cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biological activity of the cell support tissue cells is maintained.

[0041] "Substantially isolated" or "isolated" when used in the context of fetal support tissue means that the fetal support tissue has been separated from most other non-fetal support tissue material (e.g., other tissues, red blood cells, veins, arteries) derived from the original source organism.

[0042] As used herein, the phrase "wherein the biological and structural integrity of the isolated fetal support tissue is substantially preserved" means that the biological activity and structural integrity of the isolated fetal support tissue is reduced by only about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50% or about 60% compared to the biological activity and structural integrity of fresh fetal support tissue.

[0043] As used herein, "processing" refers to any activity performed on fetal support tissues or formulations containing native HC-HA / PTX3, other than recovery, donor screening, donor testing, storage, labeling, packaging or distribution, such as microbiological, viral testing, formulation, sterilization, inactivation or removal of foreign factors, preservation for storage and removal from storage. As used herein, "HC-HA / PTX3" means heavy chain 1 - hyaluronan-pentaxin 3; "-" indicates covalent attachment and " / " indicates non-covalent tight binding). HC-HA / PTX3 is present in fetal support and birth tissues, i.e., AM and UC.

[0044] As used herein, the terms "purified" and "isolated" refer to a material (e.g., a native HC-HA / PTX3 complex) that is substantially or essentially free of components that normally accompany it in its native state. In some embodiments, "purified" or "isolated" refers to a material (e.g., a native HC-HA / PTX3 complex) that is about 50% or more free of components that normally accompany it in its native state, such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% free of components that normally accompany it in its native state.

[0045] As used herein, "biological activity" refers to the activity of polypeptides and polysaccharides in fetal support tissues, including natural HC-HA / PTX3 or HA (which exists as natural HC-HA / PTX3 in fetal support tissues) or any other components of fetal support tissues and combinations thereof. In some embodiments, the biological activity of polypeptides and polysaccharides found in fetal tissue support products is anti-inflammatory, anti-scarring or anti-adhesion. In some embodiments, biological activity refers to the in vivo activity of natural HC-HA / PTX3 complexes in fetal support tissue products or the physiological response produced after in vivo administration of fetal support tissues. In some embodiments, the biological activity of natural HC-HA / PTX3 complexes in fetal support tissues is substantially preserved. In some embodiments, the activity of polypeptides and polysaccharides found in fetal tissue support products is to promote regenerative wound healing. In some embodiments, the activity of polypeptides and polysaccharides found in fetal support tissues is to prevent scarring. In some embodiments, the activity of polypeptides and polysaccharides found in fetal support tissues is to reduce inflammation. Thus, biological activity encompasses the therapeutic effects and pharmaceutical activities of fetal support tissue compositions, such as, for example, HA, native HC-HA / PTX3 complexes, and combinations thereof, or any other composition, or component of a fetal support tissue disclosed herein.

[0046] As used herein, "structural integrity" refers to the integrity of the matrix and basement membranes that make up the fetal support tissue.

[0047] As used herein, a "purified" native HC-HA / PTX3 (nHC-HA / PTX3) complex refers to a HC-HA / PTX3 complex purified from a biological source (e.g., a cell, tissue, or biological fluid). In some embodiments, a purified native HC-HA / PTX3 (nHC-HA / PTX3) complex refers to a HC-HA / PTX3 complex purified from human fetal support tissue. In some embodiments, nHC-HA / PTX3 is purified from amniotic membrane. In some embodiments, nHC-HA / PTX3 is purified from the umbilical cord. In some embodiments, the purified native HC-HA / PTX3 complex is separated and purified from the fetal support tissue before mixing with the fetal support tissue. In some embodiments, the purified native HC-HA / PTX3 complex is separated and purified from the fetal support tissue after mixing with the fetal support tissue. In some embodiments, nHC-HA / PTX3 is substantially isolated, wherein the substantial separation occurs before or after mixing with the fetal support tissue. In some embodiments, the substantially isolated HC-HA complex is derived from placenta, umbilical cord, chorion, amnion-chorion, placental amnion (PAM), umbilical cord amnion (UCAM), or any combination thereof. In some embodiments, the substantially isolated HC-HA complex is derived from frozen or formerly frozen placental amnion (PAM), frozen or formerly frozen umbilical cord amnion (UCAM), frozen or formerly frozen placenta, frozen or formerly frozen umbilical cord, frozen or formerly frozen chorion, frozen or formerly frozen amnion-chorion, or any combination thereof. In all or any cases, separation of the HC-HA complex can occur before or after mixing with the fetal support tissue.

[0048] As used herein, "hyaluronan," "hyaluronic acid," or "hyaluronate" (HA) are used interchangeably and refer to a substantially non-sulfated linear glycosaminoglycan (GAG) having repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine (D-glucuronyl-N-acetylglucosamine).

[0049] As used herein, the term "tissue with adverse changes" refers to tissue that has degenerated due to, for example, degenerative diseases (e.g., arthritis, multiple sclerosis, Parkinson's disease, muscular dystrophy, and Huntington's disease) or aging; scar tissue; or tissue that has been damaged due to insults such as burns, wounds, lacerations, injuries, ulcers, surgery, or due to ischemia.

[0050] As used herein, the term "high molecular weight" or "HMW", such as high molecular weight hyaluronic acid polysaccharide (HMW HA), refers to HA having a weight average molecular weight greater than about 500 kilodaltons (kDa), such as, for example, between about 500 kDa and about 10,000 kDa, between about 800 kDa and about 8,500 kDa, between about 1100 kDa and about 5,000 kDa, or between about 1400 kDa and about 3,500 kDa. In some embodiments, the weight average molecular weight of HMW HA is 3000 kDa or greater. In some embodiments, the weight average molecular weight of HMW HA is 3000 kDa. In some embodiments, HMW HA is The weight average molecular weight is about 3000 kDa. In some embodiments, the molecular weight of HMW HA is between about 500 kDa and about 10,000 kDa. In some embodiments, the molecular weight of HMW HA is between about 800 kDa and about 8,500 kDa. In some embodiments, the molecular weight of HMW HA is about 3,000 kDa.

[0051] As used herein, the term "low molecular weight" or "LMW", such as low molecular weight hyaluronic acid polysaccharide (LMW HA), refers to HA having a weight average molecular weight of less than 500 kDa, such as, for example, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, less than about 100 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa, less than about 20 kDa, about 200 kDa-300 kDa, about 1 kDa-300 kDa, about 15 kDa to about 40 kDa, or about 8 kDa-10 kDa.

[0052] As used herein, pentraxin 3 or PTX3 protein or polypeptide refers to any PTX3 protein, including but not limited to natural PTX3 protein and PTX3 protein extracted from cells or tissues. PTX3 includes multimeric forms (e.g., homopolymers) of PTX3, including but not limited to dimers, trimers, tetramers, pentamers, hexamers, octamers and other multimeric forms.

[0053] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of the agent or compound administered that is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent. In some embodiments, the result is to reduce and / or alleviate the signs, symptoms or causes of the disease, or any other desired biological system changes. For example, an "effective amount" for therapeutic use is an amount of a composition comprising a compound disclosed herein that provides clinically significantly reduced disease symptoms without excessive adverse side effects. In some embodiments, techniques such as dose escalation studies are used to determine the appropriate "effective amount" in any individual case. The term "therapeutically effective amount" includes, for example, a preventive effective amount. An "effective amount" of a compound disclosed herein is an amount that effectively achieves the desired effect or therapeutic improvement without excessive adverse side effects. It should be understood that in some embodiments, an "effective amount" or "therapeutically effective amount" varies from subject to subject, depending on changes in composition metabolism, subject age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. In some embodiments, an effective amount is an amount of a product or compound sufficient to promote angiogenesis or normal angiogenesis in a tissue.

[0054] As used herein, the terms "subject," "individual," and "patient" are used interchangeably. None of the terms should be interpreted as requiring supervision by a medical professional (e.g., a doctor, nurse, physician's assistant, caregiver, hospice worker). As used herein, a subject is any animal, including mammals (e.g., humans or non-human animals) and non-mammals. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0055] As used herein, the terms "treat," "treating," or "treatment," and other grammatical equivalents, include alleviating, alleviating, or ameliorating one or more symptoms of a disease or condition, ameliorating, preventing, or reducing the appearance, severity, or frequency of one or more other symptoms of a disease or condition, ameliorating or preventing the underlying metabolic causes of one or more symptoms of a disease or condition, inhibiting a disease or condition, such as arresting the development of the disease or condition, relieving a disease or condition, causing regression of a disease or condition, ameliorating a condition caused by a disease or condition, or prophylactically and / or therapeutically inhibiting symptoms of a disease or condition. In a non-limiting example, to obtain a prophylactic benefit, a native HC-HA / PTX3 complex or composition disclosed herein is administered to an individual at risk for, susceptible to, or reporting one or more physiological symptoms of a condition.

[0056] Composition

[0057] As used herein, the term "composition" refers to all combinations of two or more substances, and includes all combined products, whether chemically combined or mechanically mixed, and whether gas, fluid, powder or solid. As used herein, a "composition" of fetal support tissue can refer to any form of fetal support tissue, including isolated fetal support tissue, such as isolated HA, nHC-HA / PTX3, extracts, flake forms, non-flake forms, powders, gels, solutions, biologically active components of any form of fetal support tissue. Fetal support tissue contains a variety of innate biological factors that can be used for a variety of purposes, including wound healing and reducing inflammation and scarring. Characterization data show that the extracellular matrix (ECM) of AM and UC is rich in hyaluronic acid (HA), with an important or key matrix component called native HC-HA / PTX3 (nHC-HA / PTX3) complex (HC-HA / PTX3" represents heavy chain 1-hyaluronic acid-positive pentraxin 3; "-" represents covalent linkage, and " / " represents non-covalent tight association) is a unique extracellular matrix from birth tissue (i.e., AM and UC). Natural HC-HA / PTX3 is isolated from fetal support tissue obtained from humans or animals. Disclosed herein is a mixed fetal support tissue material containing natural HC-HA / PTX3 (nHC-HA / PTX3) complex. The nHC-HA / PTX3 complex—high molecular weight (HMW) hyaluronic acid polysaccharide (HA) covalently linked to heavy chain (HC) 1 from inter-α-trypsin inhibitor and further complexed with positive pentraxin 3 (PTX3)—is a key active component of the umbilical cord and amniotic membrane, responsible for their promotion of regenerative wound healing. The therapeutic effect of healing. Therefore, it is crucial to produce fetal support tissue products (e.g., amniotic membrane and umbilical cord extracts for wound healing) with high-yield HA complexes or nHC-HA / PTX3. The ability to produce fetal support tissue products, compositions, extracts, or combinations thereof while preventing degradation of bioactive components (e.g., HC-HA / PTX3 complexes, extracellular matrix, cytokines, growth factors, etc.) is the key to producing clinically applicable products. Due to a wide range of clinical applications, it is very important to find a method for processing fetal support tissue extracts or compositions that significantly reduces donor-to-donor variability. Disclosed herein are systems, methods, compositions, or devices for generating fetal support tissues with significantly reduced donor variability. Disclosed herein are fetal support tissues mixed from multiple donors. Mixing of fetal support tissues from multiple donors reduces donor-to-donor variability. Disclosed herein are mixed fetal support tissues processed before or after freeze drying. The freeze drying process disclosed herein results in reduced degradation of the fetal support tissue composition.Freeze-drying disclosed herein also increases the concentration of bioactive compositions, for example, increases the yield of nHC-HA / PTX3, hyaluronan polysaccharide (HA), growth factors and cytokines, other bioactive factors, while preserving the stability and potency of the fetal support tissue extract, composition or product.

[0058] Fetal support tissue is obtained from a donor after delivery. In some embodiments, the fetal support tissue may include at least 5 grams (g), 10g, 15g, 20g of amniotic membrane (wet weight) per donor; or at least 0.3 grams (g), 0.4g, 0.5g, 0.6g, 0.7g or 0.8g of dry weight (e.g., freeze-dried weight). In some embodiments, the fetal support tissue may include at least 20 grams (g), 25g, 30g, 35g, 40g, 45g, 50g of umbilical cord tissue (wet weight) per donor; or at least 0.7 grams (g), 0.8g, 0.9g, 1.0g, 1.1g or 1.2g of dry weight (e.g., freeze-dried weight). The mixed fetal support tissue described herein may be processed to separate or purify nHC-HA / PTX3 complexes.

[0059] In some embodiments, the mixed fetal support tissue disclosed herein may have a therapeutically effective amount of nHC-HA / PTX3. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is about 0.1 mg per 100 mg of mixed fetal support tissue to about 10 mg per 100 mg of mixed fetal support tissue. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is about 1 mg per 60 g of mixed fetal support tissue. For example, the therapeutically effective amount of nHC-HA / PTX3 is about 1 mg per 10 g of mixed amniotic membrane. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is about 0.1 mg per 100 mg of mixed fetal support tissue to about 0.5 mg per 100 mg of mixed fetal support tissue, about 0.1 mg per 100 mg of mixed fetal support tissue to about 1 mg per 100 mg of mixed fetal support tissue, about 0.1 mg per 100 mg of mixed fetal support tissue to about 2 mg per 100 mg of mixed fetal support tissue, about 0.1 mg per 100 mg of mixed fetal support tissue to about 3 mg per 100 mg of mixed fetal support tissue, about 0.1 mg per 100 mg of mixed fetal support tissue to about 4 mg per 100 mg of mixed fetal support tissue, about 0.1 mg per 100 mg of mixed fetal support tissue to about 10 mg per 100 mg of mixed fetal support tissue. About 5 mg, about 0.1 mg per 100 mg of mixed fetal support tissue to about 10 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue to about 1 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue to about 2 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue to about 3 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue to about 4 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue to about 5 mg per 100 mg of mixed fetal support tissue, about 0.5 mg to about 10 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue to about 2 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue to about 3 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue to about 4 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue to about 5 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue to about 10 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue to about 3 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue to about 4 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue The amount of fetal support tissue may range from about 2 mg to about 5 mg per 100 mg of mixed fetal support tissue, from about 2 mg to about 10 mg per 100 mg of mixed fetal support tissue, from about 3 mg to about 4 mg per 100 mg of mixed fetal support tissue, from about 3 mg to about 5 mg per 100 mg of mixed fetal support tissue, from about 3 mg to about 10 mg per 100 mg of mixed fetal support tissue, from about 3 mg to about 10 mg per 100 mg of mixed fetal support tissue, from about 3 mg to about 10 mg per 100 mg of mixed fetal support tissue, from about 4 mg to about 5 mg per 100 mg of mixed fetal support tissue, from about 4 mg to about 10 mg per 100 mg of mixed fetal support tissue, or from about 5 mg to about 10 mg per 100 mg of mixed fetal support tissue. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is about 0.1 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue, about 3 mg per 100 mg of mixed fetal support tissue, about 4 mg per 100 mg of mixed fetal support tissue, about 5 mg per 100 mg of mixed fetal support tissue, or about 10 mg per 100 mg of mixed fetal support tissue. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is at least about 0.1 mg per 100 mg of mixed fetal support tissue, about 0.5 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue, about 3 mg per 100 mg of mixed fetal support tissue, about 4 mg per 100 mg of mixed fetal support tissue, about 5 mg per 100 mg of mixed fetal support tissue, or about 10 mg per 100 mg of mixed fetal support tissue.5 mg, about 1 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue, about 3 mg per 100 mg of mixed fetal support tissue, about 4 mg per 100 mg of mixed fetal support tissue, or about 5 mg per 100 mg of mixed fetal support tissue. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 is up to about 0.5 mg per 100 mg of mixed fetal support tissue, about 1 mg per 100 mg of mixed fetal support tissue, about 2 mg per 100 mg of mixed fetal support tissue, about 3 mg per 100 mg of mixed fetal support tissue, about 4 mg per 100 mg of mixed fetal support tissue, about 5 mg per 100 mg of mixed fetal support tissue, or about 10 mg per 100 mg of mixed fetal support tissue. .

[0060] In some embodiments, a therapeutically effective amount of nHC-HA / PTX3 includes about 0.1 g per 10 g wet tissue to about 2 g per 10 g wet tissue. In some embodiments, the therapeutically effective amount of nHC-HA / PTX3 includes about 0.1 g per 10 g wet tissue to about 0.2 g per 10 g wet tissue, about 0.1 g per 10 g wet tissue to about 0.5 g per 10 g wet tissue, about 0.1 g per 10 g wet tissue to about 1 g per 10 g wet tissue, about 0.1 g per 10 g wet tissue to about 2 g per 10 g wet tissue, about 0.2 g per 10 g wet tissue to about 0.5 g per 10 g wet tissue, about 0.2 g per 10 g wet tissue to about 1 g per 10 g wet tissue, about 0.2 g per 10 g wet tissue to about 2 g per 10 g wet tissue, about 0.5 g per 10 g wet tissue to about 1 g per 10 g wet tissue, about 0.5 g per 10 g wet tissue to about 2 g per 10 g wet tissue, or about 1 g per 10 g wet tissue to about 2 g per 10 g wet tissue. In some embodiments, a therapeutically effective amount of nHC-HA / PTX3 includes about 0.1 g per 10 g of wet tissue, about 0.2 g per 10 g of wet tissue, about 0.5 g per 10 g of wet tissue, about 1 g per 10 g of wet tissue, or about 2 g per 10 g of wet tissue. In some embodiments, a therapeutically effective amount of nHC-HA / PTX3 includes at least about 0.1 g per 10 g of wet tissue, about 0.2 g per 10 g of wet tissue, about 0.5 g per 10 g of wet tissue, or about 1 g per 10 g of wet tissue. In some embodiments, a therapeutically effective amount of nHC-HA / PTX3 includes at most about 0.2 g per 10 g of wet tissue, at most about 0.5 g per 10 g of wet tissue, at most about 1 g per 10 g of wet tissue, or at most about 2 g per 10 g of wet tissue.

[0061] Cryopreservation

[0062] In some embodiments, the fetal support tissue is mixed and then frozen for cryopreservation. In some embodiments, the fetal support tissue is frozen for cryopreservation and mixed after freezing. In some embodiments, cryopreservation of the fetal support tissue does not destroy the integrity of the fetal support tissue extracellular matrix. In some embodiments, the fetal support tissue is exposed to liquefied gas (e.g., liquid nitrogen or liquid hydrogen). In some embodiments, the fetal support tissue is exposed to liquid nitrogen. In some embodiments, the fetal support tissue does not contact liquefied gas. In some embodiments, the fetal support tissue is placed in a container and the container is contacted with a liquefied gas. In some embodiments, the fetal support tissue is exposed to a liquefied gas until the fetal support tissue is frozen.

[0063] As used herein, "fragments" refer to tissue particles with a length, width or thickness in the range of about 0.1 mm to about 1.0 cm obtained from a larger piece of tissue. As described herein, "fragments" retain the characteristics of their source tissue and can be identified as the tissue upon inspection. As used herein, the terms "morselized", "morselizing" and "morselization" refer to the action of "fragments" related to the present application. In some embodiments, the fragmented fetal support tissue produced before or after mixing the fetal support tissue is further processed into a solution, suspension or emulsion by mixing the fragmented fetal support tissue with a carrier. In some embodiments, the fragmented fetal support tissue is formulated into a solution, suspension, paste, ointment, oily emulsion, cream, lotion, gel, patch, stick, film, coating or a combination thereof. In some embodiments, the fragmented fetal support tissue is contacted with a patch or wound dressing. In some embodiments, the fragmented fetal support tissue is formulated for parenteral injection, administered as a sterile solution, suspension, or emulsion, or formulated for inhalation.

[0064] Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue, including components of the fetal support tissue, such as nHC-HA / PTX3, HA, cytokines, growth factors, or other bioactive components. The effectiveness of treating fetal support tissue to preserve HC-HA / PTX3 in AM / UC is disclosed throughout the specification and claims. In some embodiments, fetal support tissue comprising "fragmentation" also refers to "micronization". Hereinafter, as used herein, the term "micronization" may be used interchangeably to refer to fragmentation, grinding, chopping, stirring, mixing, pulverizing, powdering, homogenization, and any combination thereof of fetal support tissue. In some embodiments, the fetal support tissue may include amniotic tissue and umbilical cord tissue mixed in any ratio of 0.001:99.999 w / w% to 99.999:0.001 w / w%, micronized before mixing and / or freeze drying the fetal support tissue or after mixing and / or freeze drying the fetal support tissue obtained from fresh or frozen tissue using any micronizing tool known to those skilled in the art, such as, for example, a tissue grinder, sonicator, bread mixer, freezer / mill, blender, mortar / pestle, rotary stator, kitchen chopper, grinder, ruler and scalpel to produce fragments with a length, width or thickness ranging from about 0.1 mm to about 1.0 cm in size. In some embodiments, the resulting fragments are micronized to produce particles of uniform size. In some embodiments, the resulting fragments are used in a wet, partially dehydrated or substantially dehydrated state, and the dehydration method is any method known to those skilled in the art, such as, for example, centrifugation, ultracentrifugation or freeze drying. In some embodiments, the resulting fetal support tissue is used immediately or stored in any type of container known to those skilled in the art (e.g., bags, jars, bottles, tubes, ampoules, and pre-filled syringes) for later use. In some embodiments, the micronized fetal support tissue is sterilized by any method known to those skilled in the art (e.g., gamma irradiation).

[0065] As used herein, "comminuted fetal support tissue" refers to fetal support tissue that contains "micronized" tissue that has been broken (or separated), and in some cases, crushing and micronization are used interchangeably. In some embodiments, the crushed mixed fetal support tissue is a dry powder. In some embodiments, the crushed mixed and / or freeze-dried fetal support tissue is a dry powder mixed after micronization. In some embodiments, the separated fetal support tissue is used to generate micronized fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue before mixing the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue after mixing the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue before mixing the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue before freeze-drying the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue before or after freeze-drying the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue before mixing the fetal support tissue but before or after freeze drying the fetal support tissue. In some embodiments, the fetal support tissue is used to generate micronized fetal support tissue after mixing the fetal support tissue but before or after freeze drying the fetal support tissue. In some embodiments, the micronized fetal support tissue processed before or after mixing the fetal support tissue and before or after freeze drying the fetal support tissue is further processed into a solution, suspension or emulsion by mixing the fetal support tissue powder with a carrier. In some embodiments, the micronized fetal support tissue (produced before or after mixing, and / or before or after freeze drying and / or in a combination of the two) is formulated into a solution, suspension, paste, ointment, oily emulsion, cream, lotion, gel, patch, stick, film, coating or a combination thereof. In some embodiments, the micronized fetal support tissue is contacted with a patch or wound dressing. In some embodiments, the micronized fetal support tissue is formulated for parenteral injection, administered with a sterile solution, suspension or emulsion, or formulated for inhalation.

[0066] In certain embodiments, disclosed herein are methods and processes for generating fetal support tissue while preserving the biological activity of HC-HA / PTX3 from fetal support tissue. In certain embodiments, the process includes a mixing step that reduces the variability of the amount or activity of HC-HA / PTX3. In some embodiments, the separated fetal support tissue is micronized by any suitable method. In some embodiments, the freeze drying of the fetal support tissue occurs before the mixed fetal support tissue is micronized. In some embodiments, the freeze drying of the fetal support tissue may occur after the mixed fetal support tissue is micronized. In some embodiments, the separated fetal support tissue is micronized by using a pulverizer (e.g., Bessman tissue pulverizer, Biospec biological pulverizer, or Covaris CryoPrep). In some embodiments, the separated fetal support tissue is micronized by using a tissue grinder (e.g., Potter-Elvehjem grinder or Wheaton overhead stirrer). In some embodiments, the separated fetal support tissue is micronized by using an ultrasonicator. In some embodiments, the separated fetal support tissue is micronized by using a bead mill. In some embodiments, the isolated fetal support tissue is prepared by using a freezer / mill (e.g., In some embodiments, the isolated fetal support tissue is micronized using a pestle and mortar. In some embodiments, the isolated fetal support tissue is micronized manually using a pestle and mortar. Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims).

[0067] Disclosed herein is a process for generating fetal support tissue while preserving the biological activity of fetal support tissue (including components of fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims). In some embodiments, the fetal support tissue can be in the form of flakes or non-flakes of flake products of different sizes. In some embodiments, the mixed fetal support tissue is micronized before freezing the fetal support tissue. In some embodiments, the mixed fetal support tissue is micronized after freezing the fetal support tissue. In some embodiments, the fetal support tissue is not mixed before micronization. In some embodiments, the fetal support tissue is mixed after micronization. In some embodiments, the fetal support tissue is micronized before freeze drying and before or after mixing. In some embodiments, the fetal support tissue is micronized after freeze drying and before or after mixing. In some embodiments, the mixed fetal support tissue is freeze dried before micronization, inactivation, decellularization, cryo-crushing, sterilization, cryopreservation, purification of dry or wet fetal support tissue extracts, or a combination thereof. In some embodiments, the mixed fetal support tissue is freeze-dried after micronization, sieving, centrifugation, filtration, inactivation, decellularization, cryo-crushing, sterilization, cryopreservation, purification of dry or wet fetal support tissue extracts, or a combination thereof. In some embodiments, the unmixed fetal support tissue is freeze-dried before sieving, micronization, inactivation, decellularization, cryo-crushing, sterilization, cryopreservation, purification of dry or wet fetal support tissue extracts, or a combination thereof. In some embodiments, the unmixed fetal support tissue is not freeze-dried before micronization, inactivation, decellularization, cryo-crushing, sterilization, sieving, centrifugation, filtration, cryopreservation, purification of dry or wet fetal support tissue extracts, or a combination thereof. In some embodiments, micronization, inactivation, decellularization of dry or wet tissues, cryo-crushing, sterilization, cryopreservation, purification, or a combination thereof occurs before mixing the fetal support tissue, wherein the fetal support tissue can be fresh, or frozen, or unfrozen, or previously frozen, or being frozen. In some embodiments, micronization, inactivation, decellularization of dry or wet tissues, cryo-crushing, sterilization, cryopreservation, purification, or a combination thereof occurs on mixed fetal support tissue (i.e., after mixing), and is generated from mixed fresh fetal support tissue, mixed frozen fetal support tissue, mixed and then frozen fetal support tissue, or mixed fetal support tissue immediately before freezing (decellularization), or from mixed tissue after freezing (decellularization) of fetal support tissue. In some embodiments, mixed fetal support tissue can be used to produce an extract, and native HC-HA / PTX3 complex (nHC-HA / PTX3) can be purified from the extract by any suitable method. In some embodiments, the mixing strategies described herein can be used for manufacturing by purifying nHC-HA / PTX3.In some embodiments, the purified mixed fetal support tissue product can be any form of extract, composition, or product disclosed herein, wherein the product or extract is purified by centrifugation. Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims).

[0068] Various forms of fetal support tissue

[0069] In some embodiments, the fetal support tissue includes micronized fetal support tissue. In some embodiments, the fetal support tissue can be a fetal support tissue transplant, a fetal support tissue slice, a fetal support tissue homogenate, a fetal support tissue extract. In some embodiments, the fetal support tissue is freeze-dried. In some embodiments, the fetal support tissue is centrifuged or ultracentrifuged. In some embodiments, the fetal support tissue is liquid. In some embodiments, the liquid fetal support tissue is filtered. In some embodiments, the fetal support tissue is powder. In some embodiments, the powdered fetal support tissue is sieved. In some embodiments, the fetal support tissue is inactivated or decellularized. In some embodiments, the fetal support tissue is terminally sterilized. In some embodiments, the fetal support tissue is a purified natural HC-HA / PTX3 complex. In some embodiments, the fetal support tissue is an extract of fetal support tissue. In some embodiments, the fetal support tissue is placental amnion, umbilical cord, umbilical cord amnion, chorion, amnion-chorion, placenta, amniotic matrix, amniotic gel or any combination thereof.

[0070] In some embodiments, the fetal support tissue comprises the umbilical cord, the amnion, or the umbilical cord amnion. In some embodiments, the umbilical cord comprises the umbilical cord, the amnion, and at least some Wharton's jelly. In some embodiments, the umbilical cord comprises the umbilical cord, the amnion, and the Wharton's jelly. In some embodiments, the umbilical cord comprises the umbilical cord, the amnion, and is substantially free of Wharton's jelly. In some embodiments, the umbilical cord lacks the umbilical cord vein and artery. In some embodiments, the umbilical cord comprises the umbilical cord vein and artery.

[0071] In some embodiments, the fetal support tissue includes an extract of the fetal support tissue. In some embodiments, the fetal support tissue includes a natural HC-HA / PTX3 complex (nHC-HA / PTX3). In some embodiments, the fetal support tissue consists essentially of nHC-HA / PTX3. In some embodiments, the fetal support tissue includes other components, such as, for example, other extracellular matrices, growth factors, cytokines, and other biologically active components that can be separated from the fetal support tissue. Disclosed herein is a process for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as, for example, HA, HC-HA / PTX3 growth factors, cytokines, and other active biological components), wherein the effectiveness of HC-HA / PTX3 can be monitored to ensure that it is fully preserved, as disclosed in the specification and claims. In certain embodiments, HC-HA / PTX3 is biologically active.

[0072] Grinding / freeze grinding

[0073] In some embodiments, the fetal support tissue is ground by any suitable method before or after mixing and before or after freeze drying. In some embodiments, the mixing of the fetal support tissue before or after freeze drying occurs on fresh fetal support tissue, original fetal support tissue, newly frozen fetal support tissue, or once frozen fetal support tissue. Exemplary procedures for micronizing or freezing and crushing fetal support tissue are disclosed herein. In some embodiments, the fetal support tissue is mixed before micronizing or before freezing and crushing. In some embodiments, the fetal support tissue is mixed after micronizing or after freezing and crushing. In some embodiments, grinding the fetal support tissue includes freezing and crushing the fetal support tissue. In some embodiments, freezing and crushing the fetal support tissue includes crushing, micronizing or otherwise breaking the fetal support tissue when the fetal support tissue is in a frozen state (e.g., exposed to temperatures below 0°C, -20°C, -40°C, -50°C, -60°C, -70°C, -75°C, -80°C, -90°C, -100°C) or an icy state. In some embodiments, the frozen pulverized fetal support tissue includes micronizing the fetal support tissue in a cryogenically controlled environment. In some embodiments, the frozen pulverized fetal support tissue includes micronizing the fetal support tissue after immersing or exposing the fetal support tissue to (e.g., directly or indirectly) liquid nitrogen. In some embodiments, the frozen pulverized fetal support tissue includes micronizing the fetal support tissue while immersing or exposing the fetal support tissue to (e.g., directly or indirectly) liquid nitrogen. In some embodiments, the frozen pulverized fetal support tissue includes placing the fetal support tissue in a grinding container and immersing the grinding container in liquid nitrogen before grinding. In some embodiments, the grinding container is immersed in liquid nitrogen for at least 1 minute during the grinding process. In some embodiments, the frozen pulverized fetal support tissue includes exposing the frozen fetal support tissue to a hammer or a rotating blade. In some embodiments, the frozen pulverized fetal support tissue includes exposing the frozen fetal support tissue to an impactor. In some embodiments, the impactor is driven by an electromagnet. In some embodiments, the fetal support tissue is frozen and pulverized by using a cryomill. In some embodiments, the fetal support tissue is frozen and pulverized by using a mortar and pestle. In some embodiments, the fetal support tissue is frozen and pulverized by using a blender. In some embodiments, the fetal support tissue is cryo-pulverized using a bio-pulverizer. In some embodiments, cryo-pulverizing the fetal support tissue in liquid nitrogen avoids activating fetal support tissue proteases and / or hyaluronidases that can degrade proteins and hyaluronic acid polysaccharides in the fetal support tissue, compared to grinding unfrozen fetal support tissue.Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue, including components of the fetal support tissue, such as HA, nHC-HA / PTX3, growth factors, cytokines, and other active biological components, wherein the potency of the HC-HA / PTX3 can be monitored to ensure its adequate preservation, as disclosed in the specification and claims.

[0074] In some embodiments, cryopreservation reduces fetal support tissue to powder. In some embodiments, the particles comprising powder have a uniform size distribution. In some embodiments, the particles comprising powder have a non-uniform size distribution. In some embodiments, fetal support tissue is reduced to a particle size of less than about 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm or 0.1 μm. In some embodiments, fetal support tissue is reduced to a particle size of less than 500 μm. In some embodiments, fetal support tissue is reduced to a particle size of less than about 0.5 μm. In some embodiments, fetal support tissue is reduced to a particle size of less than 0.3 μm.

[0075] Freeze drying

[0076] Disclosed herein is a process for generating fetal support tissue while preserving the biological activity of fetal support tissue (including components of fetal support tissue, such as nHC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims). In some embodiments, freeze drying preserves the biological activity of HC-HA / PTX3. In some cases, the cryopreserved fetal support tissue or tissue product is placed in a vacuum chamber of a freeze drying device until all or substantially all fluids (e.g., water) are removed. In some embodiments, the fetal support tissue is cryopreserved before or after mixing. In certain embodiments, the water content of the mixed fetal support tissue is reduced to 20% or less, 15% or less, 10% or less, or 5% or less by weight. In certain embodiments, the water content of the mixed fetal support tissue is 1% or more, 2% or more, 3% or more, 5% or more, or 10% or more by weight. In some embodiments, the fetal support tissue from an individual and / or a small mix or donor (e.g., 2, 3, 4, or 5) is freeze dried before mixing. In some embodiments, the mixed fetal support tissue is freeze-dried after mixing. In some embodiments, the mixed fetal support tissue is fresh, or newly frozen, or has been frozen and stored before or after freeze drying. In some embodiments, the mixed fetal support tissue is freeze-dried, and then micronized, sieved, cryopreserved, sterilized, purified, centrifuged, filtered, or any combination thereof to obtain products of different sizes. In some embodiments, the fetal support tissue is freeze-dried before mixing. In some embodiments, the fetal support tissue is not freeze-dried before mixing. In some embodiments, the fetal support tissue is micronized, sieved, or cryopreserved, sterilized, purified, centrifuged, filtered, or a combination thereof before or after freeze drying to obtain products of different sizes. In some embodiments, the fetal support tissue is freeze-dried and then mixed after micronization, sieving, or cryopreservation, sterilization, purification, centrifugation, filtration, or a combination thereof to obtain products of different sizes. In some embodiments, the mixed fetal support tissue is freeze-dried after freezing. In some embodiments, the fetal support tissue is freeze-dried by any suitable method (eg, exposure to liquefied gas, placement in a freezer) after freezing and then mixed.

[0077] As disclosed herein, in some embodiments, the fetal support tissue is mixed fetal support tissue, wherein the fetal support tissue is mixed before or after freeze-drying, wherein the fetal support tissue is mixed before or after micronization, sieving, or cryo-crushing, cryopreservation, centrifugation, filtration, sterilization, decellularization, inactivation, freezing, thawing, cutting, cleaning, or any other processing disclosed herein and / or combinations thereof.

[0078] In some embodiments, the mixed fetal support tissue is frozen by being exposed to a temperature below about 0°C. In some embodiments, the mixed fetal support tissue is frozen by being exposed to a temperature below about -20°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -40°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -50°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -60°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -70°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -75°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -80°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -90°C. In some embodiments, the fetal support tissue is frozen by being exposed to a temperature below about -100°C. In some embodiments, the fetal support tissue is frozen by being exposed to a liquefied gas. For example, liquid nitrogen gas. In some embodiments, the fetal support tissue is placed in a vacuum chamber of a freeze drying apparatus until all or substantially all fluid (eg, water) is removed. In some embodiments, the cryopreserved fetal support tissue is freeze dried.

[0079] Production of fetal support tissue

[0080] In some embodiments, the fetal support tissue is derived from umbilical cord (UC) tissue. In some embodiments, the fetal support tissue is derived from amniotic membrane (AM) tissue. In some embodiments, the fetal support tissue is derived from umbilical cord amniotic membrane tissue or any combination thereof. In some embodiments, the fetal support tissue includes a fetal support tissue separated without veins or arteries. In some embodiments, the fetal support tissue includes a fetal support tissue separated without veins or arteries and metabolically active cells, wherein the natural structural integrity of the fetal support tissue is substantially preserved after initial purchase. In some embodiments, the fetal support tissue includes a fetal support tissue separated including veins or arteries. In some embodiments, the fetal support tissue includes a fetal support tissue separated including veins or arteries and metabolically active cells, wherein the natural structural integrity of the fetal support tissue is substantially preserved after initial purchase. In some embodiments, the fetal support tissue includes umbilical cord amniotic membrane and Wharton's jelly. In some embodiments, the biological activity of various bioactive components (e.g., HA, nHC-HA / PTX3 complexes or compositions) in the fetal support tissue is substantially preserved. In some embodiments, the biological activity of the bioactive components, HA and / or nHC-HA / PTX3 complexes in the fetal support tissue is substantially preserved when processed (washed, cut, centrifuged, etc.) and frozen 72 hours after delivery. The frozen tissue can be frozen for at least 1 day, 5 days, 10 days, 20 days, 30 days or longer. In some embodiments, the biological activity of the bioactive components, HA or nHC-HA / PTX3 complexes in the fetal support tissue is substantially preserved when it is processed (washed, cut, etc.) and frozen no more than 72 hours after delivery. In some embodiments, the biological activity of the bioactive components, HA or nHC-HA / PTX3 complexes in the fetal support tissue is substantially preserved for at least 15 days. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 20 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 25 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 30 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 35 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 40 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 45 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 50 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 55 days after initial purchase.In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 60 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 65 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 70 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 75 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 80 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 85 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 90 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 95 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 100 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 180 days after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 1 year after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 2 years after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 3 years after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 4 years after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for at least 5 years after initial purchase. In some embodiments, the biological and structural integrity of the fetal support tissue is substantially preserved for one or more days to at least 5 years after initial purchase. In some embodiments, the fetal support tissue is obtained from an animal, a mammal (e.g., a human), or a non-human animal (e.g., a primate, a cow, or a pig).

[0081] In some embodiments, the fetal support tissue is kept below 0°C until the qualifications of the donor and the sample are determined. In some embodiments, the fetal support tissue is kept between 0°C and -80°C until the qualifications of the donor and the sample are determined. In some embodiments, storing the fetal support tissue at -80°C will kill substantially all cells found in the fetal support tissue. In some embodiments, storing the fetal support tissue at -80°C will kill substantially all cells found in the fetal support tissue, while maintaining or increasing the biological activity of the fetal support tissue relative to fresh (ie, non-frozen) fetal support tissue (e.g., its anti-inflammatory, anti-scar, anti-antigen and anti-adhesion properties). In some embodiments, storing the fetal support tissue at -80°C will result in the loss of metabolic activity of substantially all cells found in the fetal support tissue. In some embodiments, the loss of metabolic activity can lead to cell death. In some embodiments, the cell may lack metabolic activity while maintaining the biological activity of the fetal support tissue or any biological molecule present therein (including but not limited to HC-HA / PTX3). In some embodiments, the fetal support tissue is dry. In some embodiments, the fetal support tissue is not dehydrated. Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims).

[0082] Processing of fetal support tissue

[0083] This article and the entire disclosure disclose Figure 1A-1B and Figure 2A , Figure 2B and Figure 2C Whenever fetal support tissue is disclosed herein, it is understood, although not limiting, that the fetal support tissue can be mixed fetal support tissue, where mixing occurs prior to the specified fetal support tissue processing steps, or can be mixed fetal support tissue, where mixing occurs prior to the specified fetal support tissue processing steps. Figure 1A-1B and Figure 2A-2B and Figure 2C After the prescribed fetal support tissue processing steps are shown.

[0084] In some embodiments, processing is performed in accordance with Good Tissue Practice (GTP) to prevent the introduction, transmission, contamination, or cross-contamination of potential infectious diseases from the donor.

[0085] In some embodiments, fetal support tissue is tested for active viral infection using FDA-approved screening tests, such as, for example, active HIV-1, HIV-2, HTLV-1, hepatitis B and C, West Nile virus, cytomegalovirus, and Treponema pallidum. In some embodiments, any sign that the tissue is contaminated with active viral infection (e.g., HIV-1, HIV-2, HTLV-1, hepatitis B and C, West Nile virus, or cytomegalovirus) will result in immediate isolation and subsequent destruction of the tissue sample. In some embodiments, the donor's medical records are checked to find risk factors and clinical evidence of hepatitis B, hepatitis C, or HIV infection.

[0086] In some embodiments, the fetal support tissue is frozen prior to mixing. In some embodiments, the fetal support tissue is frozen after mixing. In some embodiments, the mixed fetal support tissue is not frozen. In some embodiments, the unfrozen fetal support tissue is not mixed. If the fetal support tissue is not frozen, it is mixed first and then processed as described below immediately before freezing. In some embodiments, if the fetal support tissue is not frozen, it is processed as described below immediately before mixing or freezing.

[0087] In some embodiments, substantially all of the blood (e.g., from any arteries and veins found in the fetal support tissue and blood that has infiltrated the tissue) is removed from the fetal support tissue prior to mixing. In some embodiments, substantially all of the blood is removed prior to mixing and before the fetal support tissue is frozen. In some embodiments, substantially all of the blood is removed prior to mixing and after the fetal support tissue is frozen. In some embodiments, substantially all of the blood (e.g., from any arteries and veins found in the fetal support tissue or blood that has infiltrated the tissue) is not removed from the fetal support tissue prior to mixing. In some embodiments, mixing of the fetal support tissue occurs after substantially all of the blood is removed from the fetal support tissue. In some embodiments, mixing of the fetal support tissue occurs before substantially all of the blood is removed from the fetal support tissue. For example, blood is not removed prior to mixing and before the fetal support tissue is frozen. In some embodiments, substantially all of the blood is not removed prior to mixing or after the fetal support tissue is frozen. In some embodiments, blood is removed from the fetal support tissue prior to mixing. In some embodiments, blood is not removed from the fetal support tissue prior to mixing. In some embodiments, blood is removed from the fetal support tissue after mixing. In some embodiments, blood is not removed from the fetal support tissue after mixing. In some embodiments, blood is not removed from the fetal support tissue, and the fetal support tissue can be mixed after the blood is removed. In some embodiments, blood is removed from the fetal support tissue before mixing the fetal support tissue but before freezing the fetal support tissue. In some embodiments, blood is not removed from the fetal support tissue after mixing the fetal support tissue and before freezing the fetal support tissue. In some embodiments, blood is substantially removed before mixing the fetal support tissue, after the fetal support tissue has been frozen. In some embodiments, blood is substantially removed after mixing the fetal support tissue, after the fetal support tissue has been frozen.

[0088] In some embodiments, the fetal support tissue is washed with a buffer before mixing and optionally stirred to remove excess blood and tissue. In some embodiments, after mixing the fetal support tissue, the fetal support tissue is washed with a buffer and stirred to remove excess blood and tissue. In some embodiments, the fetal support tissue is first mixed before stirring with a buffer to remove excess blood. In some embodiments, before mixing, the fetal support tissue is not soaked in a buffer and stirred to remove excess blood and tissue. In some embodiments, after mixing the fetal support tissue, the fetal support tissue is not soaked in a buffer and stirred to remove excess blood and tissue.

[0089] In some embodiments, the fetal support tissue is a fetal support tissue transplant. In some embodiments, the fetal support tissue is separated and used to generate a fetal support tissue transplant before mixing. In some embodiments, the fetal support tissue is cut into multiple blocks (e.g., using a scalpel). The size of these blocks depends on the intended use of the fetal support tissue transplant derived from the fetal support tissue. In some embodiments, the cut fetal support tissue is optionally cleaned again with a buffer to further remove excess blood and tissue.

[0090] The umbilical cord (UC) includes two arteries (the umbilical arteries) and one vein (the umbilical vein). In some embodiments, the vein and artery are removed from the UC. In some embodiments, the vein and artery are not removed from the UC. In some cases, the vein and artery are surrounded (or suspended or buried) in Wharton's jelly. In some embodiments, the vein and artery are removed simultaneously with a portion of Wharton's jelly.

[0091] The desired thickness of the fetal support tissue determines how to handle the fetal support tissue. In some embodiments, the desired thickness of the fetal support tissue determines how much Wharton's jelly is removed. In some embodiments, the fetal support tissue is contacted with a buffer to promote the separation of Wharton's jelly and the umbilical cord amniotic membrane. In some embodiments, Wharton's jelly is removed using a peeling, a rotary cutter (i.e., a catheter connected to a drill bit, the drill bit is equipped with a diamond-coated grinding head), liposuction, a high-pressure liquid, a brush (e.g., a mechanical brush rotating at high speed) or a surgical skin remover. In some embodiments, Wharton's jelly is not removed from the fetal support tissue. In some embodiments, Wharton's jelly and the umbilical vein and artery are not removed. In some embodiments, Wharton's jelly is not removed, and the umbilical vein and artery are removed from the fetal support tissue before or after mixing.

[0092] In some embodiments, the fetal support tissue comprises isolated umbilical cord amniotic membrane (UCAM). Disclosed herein is a process for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims). In some embodiments, UCAM is separated by partially removing some Wharton's jelly and umbilical blood vessels from UC, leaving UCAM. After obtaining substantially pure UCAM, UCAM is optionally washed with a buffer to remove excess blood and tissue. In some embodiments, UCAM comprises Wharton's jelly. In some embodiments, UCAM comprises Wharton's jelly and umbilical veins and arteries. In some embodiments, UCAM comprises Wharton's jelly but does not include umbilical veins and arteries.

[0093] In some embodiments, the fetal support tissue has any suitable shape (e.g., square, circular, triangular, rectangular). In some embodiments, the fetal support tissue is generated from a fetal support tissue sheet. In some embodiments, the sheet is flat. In some embodiments, the sheet is tubular.

[0094] In some embodiments, the fetal support tissue is cut into multiple blocks (e.g., using a scalpel). In some embodiments, the fetal support tissue is divided into blocks of about 1.0 cm × about 0.25 cm, 0.5 cm, 0.75 cm, 1.0 cm, 2.0 cm, 3.0 cm, 4.0 cm, 5.0 cm or 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 2 cm × about 2 cm, 3 cm, 4 cm, 5 cm or 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 3 cm × about 3 cm, 4 cm, 5 cm or 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 4 cm × about 4 cm, 5 cm or 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 5 cm × about 5 cm or 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 6 cm × about 6 cm. In some embodiments, the fetal support tissue is divided into blocks of about 8 cm x about 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, or 8 cm. In some embodiments, the fetal support tissue is divided into blocks of about 10 cm x about 10 cm. In some embodiments, the fetal support tissue is divided into blocks of about 12 cm x about 10 cm. In some embodiments, the fetal support tissue is divided into blocks of about 15 cm x about 10 cm. In some embodiments, the fetal support tissue is divided into blocks of about 20 cm x about 10 cm. In some embodiments, the fetal support tissue is divided into blocks of about 25 cm x about 10 cm. In some embodiments, the fetal support tissue is divided into blocks of about 30 cm x about 10 cm.

[0095] In some embodiments, the fetal support tissue is contacted with a buffer under agitation to remove substantially all remaining red blood cells. In some embodiments, the fetal support tissue is contacted with the buffer for 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, or more than 24 hours. In some embodiments, the UC product is contacted with the buffer for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. The fetal support tissue can be washed 1x, 2x, 3x, 4x, 5x, or more. The fetal support tissue may be translucent with no visible blood stains after observation.

[0096] Mixed multiple donors

[0097] In certain embodiments, there is provided herein a method for producing a mixed fetal support tissue product. The processing of mixed fetal support tissue results in a reduction in sample-to-sample variability as seen in a single donor product and due to the high variability in a single donor product. It is difficult to implement, develop control strategies and verify efficacy determinations for single donor products to ensure consistency. However, mixed donor samples show an increased HA protein content, reduce sample-to-sample variability, and improve sample uniformity, which indicates that the mixing of donor fetal support tissue increases the consistency and uniformity of the fetal support tissue product or extract generated. In some embodiments, mixed fetal support tissue results in a reduction in sample-to-sample variability. For example, the standard deviation or percentage coefficient of variation obtained after processing a single donor sample may be higher than the standard deviation or percentage coefficient of variation obtained after processing a mixed sample. In some embodiments, the variation between a single donor sample process and a mixed donor tissue sample process may be statistically significant. Compared with the variation after processing a mixed tissue sample, the variation obtained after processing a single donor sample may be higher by an order of magnitude. For example, the determination performed using a mixed donor sample can have a variability reduction of at least about 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7 or more than the determination performed using a single donor sample. As disclosed herein, mixing fetal tissue samples from multiple donors or multiple donors allows for the expansion of production processing and manufacturing scale without sacrificing product efficacy, consistency, stability, uniformity or purity, while also allowing for effective assessment and management of quality control processing. In some embodiments, disclosed herein is mixing fetal support tissue at various stages of processing fetal support tissue, while using parameters recommended for large-scale processing and commercialization. The mixing of fetal support tissue products occurs at each step of production to significantly eliminate problems that can occur during single donor manufacturing and processing (some of which are as described above and are known in the art).

[0098] In some embodiments, the amount of HA obtained from the mixed fetal support tissue may be, on average, higher than the average HA obtained from a collection of single donor samples. For example, the amount of HA in two or more pooled donor samples can range from at least 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, 170 μg / ml, 180 μg / ml, 190 μg / ml, 200 μg / ml, 210 μg / ml, 220 μg / ml, 230 μg / ml, 240 μg / ml, 250 μg / ml, 260 μg / ml, 270 μg / ml, 280 μg / ml, 290 μg / ml ml, 300μg / ml, 310μg / ml, 320μg / ml, 330μg / ml, 340μg / ml, 350μg / ml, 360μg / ml, 370μg / ml, 380μg / ml, 390μg / ml, 400μg / ml, 410μg / ml, 420μg / ml, 430μg / ml, 440μg / ml, 450μ g / ml, 460μg / ml, 470μg / ml, 480μg / ml, 490μg / ml to about 500μg / ml, 510μg / ml, 520μg / ml, 53 0μg / ml, 540μg / ml, 550μg / ml, 560μg / ml, 570μg / ml, 580μg / ml, 590μg / ml to about 600μg / ml.In some embodiments, the amount of protein obtained from two or more pooled donor samples can be at least 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / ml, 100 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, 170 μg / ml , 180μg / ml, 190μg / ml, 200μg / ml, 220μg / ml, 230μg / ml, 240μg / ml, 250μg / ml, 260μg / ml, 270μ g / ml, 280μg / ml, 290μg / ml, 300μg / ml, 310μg / ml, 320μg / ml, 330μg / ml, 340μg / ml, 350μg / ml, 3 60μg / ml, 370μg / ml, 380μg / ml, 390μg / ml, 400μg / ml, 410μg / ml, 420μg / ml, 430μg / ml, 440μg / ml, 450μg / ml, 460μg / ml, 470μg / ml, 480μg / ml, 490μg / ml to about 500μg / ml, 510μg / ml, 520μg / ml, 5 30μg / ml, 540μg / ml, 550μg / ml, 560μg / ml, 570μg / ml, 580μg / ml, 590μg / ml, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 850μg / ml, 900μg / ml, 950μg / ml, 1000μg / ml to about 2000μg / ml.

[0099] In some embodiments, the mixing of fetal support tissue increases the amount of protein content generated from the fetal support tissue. In some embodiments, the amount of total protein obtained from the mixed fetal support tissue can be, on average, higher than the average total protein from a single donor. In some embodiments, the amount of total protein obtained from two or more mixed donor samples can be about 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / ml, 100 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, 170 μg / ml, 180 μg / ml, 190μg / ml, 200μg / ml, 220μg / ml, 230μg / ml, 240μg / ml, 250μg / ml, 260μg / ml, 270μg / ml, 2 80μg / ml, 290μg / ml, 300μg / ml, 310μg / ml, 320μg / ml, 330μg / ml, 340μg / ml, 350μg / ml, 360μg / ml , 370μg / ml, 380μg / ml, 390μg / ml, 400μg / ml, 410μg / ml, 420μg / ml, 430μg / ml, 440μg / ml, 450μg / ml, 460μg / ml, 470μg / ml, 480μg / ml, 490μg / ml to about 500μg / ml, 510μg / ml, 520μg / ml, 530μg / ml, 540 2000 μg / ml, 550 μg / ml, 560 μg / ml, 570 μg / ml, 580 μg / ml, 590 μg / ml, 600 μg / ml, 650 μg / ml, 700 μg / ml, 750 μg / ml, 800 μg / ml, 850 μg / ml, 900 μg / ml, 950 μg / ml, 1000 μg / ml to above 2000 μg / ml, depending on the number of donor samples mixed.

[0100] In some cases, the method for producing mixed fetal support tissue includes any method and mixing step for producing fetal support tissue products described herein. In some cases, the mixing step includes mixing fetal support tissues derived from multiple donors. In some cases, the mixing step includes mixing fetal support tissue products derived from multiple donors to produce a mixed composition (e.g., a mixed bulk drug). In some cases, the fetal support tissue product is produced by the method described herein, which may include the following treatments: (a) micronizing or freezing and crushing fetal support tissue to generate micronized or frozen and crushed fetal support tissue; (b) extracting micronized or frozen and crushed fetal support tissue in an excipient to generate an extract; and (c) sterilizing the extract by filtering (liquid extract) or sieving (powder extract) to produce a fetal support tissue product. In some embodiments, the processing (a) of fetal support tissue may occur before or after mixing fetal support tissue. In some embodiments, the processing (a) of fetal support tissue may occur before freeze drying of fetal support tissue. In some embodiments, the processing (a) of fetal support tissue may occur after freeze drying of fetal support tissue. In some embodiments, the freeze drying of fetal support tissue can occur before or after micronization or cryo-crushing; or it can occur before or after the mixing of donor fetal support tissue. In some cases, the donor batch is an input placenta or any other fetal support tissue material from a single donor. In some cases, the mixing step includes mixing fetal support tissue products derived from multiple donor batches to produce a mixed composition (e.g., mixed bulk drugs). In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from (e.g., derived from) at least 15 donors to produce a mixed composition. In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from at least 30 donors to produce a mixed composition. In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from at least 45 donors to produce a mixed composition. In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 donors to produce a mixed composition. In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from up to 5 donors. In some cases, the mixing step includes mixing fetal support tissues (e.g., fetal support tissue products) from up to 9, 8, 7, 6, 5, 4, 3 or 2 donors. In some cases, the mixing step includes mixing fetal support tissue products from 2 or more donors.In some cases, the mixing step includes mixing multiple batches of fetal support tissue products to produce a mixed composition. In some embodiments, the mixing step includes mixing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 batches. In some embodiments, mixing is no more than three batches to produce a mixed composition. In some embodiments, batch is a fetal support tissue composition mixed from a fetal support tissue product derived from multiple donors. In some cases, batch can include a fetal support tissue product derived from 15 donors. In some embodiments, batch is mixed by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 donors. In some cases, batch is mixed by up to 9, 8, 7, 6, 5, 4, 3 or 2 donors. In some cases, mixing includes mixing three batches, and each batch includes fetal support tissue from 15 donors. In the step of mixing fetal support tissue described herein, mixing can be performed before or after freeze drying the fetal support tissue, wherein freeze drying of the fetal support tissue occurs before or after mixing.

[0101] When to mix

[0102] Disclosed herein is a description of an exemplary procedure showing the processing and mixing times of fetal support tissue materials processed and manufactured under commercial production conditions ( Figure 1A-1B and Figure 2A-2C ).like Figure 1A-1B and Figure 2A-2C As shown, potential donors are screened; raw materials (fetal support tissue) are collected from donors and shipped to a processing plant for receipt and inspection control to determine donor qualifications. In some embodiments, the biological sample is frozen at -80°C prior to mixing or processing by cleaning, cutting and soaking in 0.9% saline. In some embodiments, the processing of the biological sample is performed by cleaning, cutting and soaking in 0.9% saline before freezing. Exemplary steps or procedures for processing and mixing of fetal support tissue (including parameters and in-process controls and critical quality attributes) are as follows Figure 1A-1B and Figure 2A-2C Once the evaluation of the listed critical quality attributes is completed, the biological sample (fetal support tissue material) will become a qualified starting material (RSM).

[0103] In some embodiments, the qualified starting material (RSM) or fetal support tissue material is mixed and processed to generate a flake form or non-flake form product. In some embodiments, the qualified starting material or fetal support tissue material is processed into a flake form product and a non-flake form product before mixing. In some embodiments, the mixed fetal support tissue is processed by micronization. In some embodiments, micronization is used to generate a gel product. In some embodiments, the mixed fetal support tissue that is micronized into a dry powder and liquefied can be further filtered before or after terminal sterilization. In some embodiments, the micronized fetal support tissue in liquid form can be centrifuged before or after terminal sterilization (e.g., using γ sterilization or a combination thereof). In some embodiments, filtration controls the sterilization of the fetal support tissue extract in liquid form, wherein the filter pore size used is equal to or approximately less than 0.2 μm. In some embodiments, the dry powder form formed by micronization of the fetal support tissue is not liquefied, but is sieved and used immediately or frozen at -80°C. In some embodiments, the micronization and sieving of the fetal support tissue extract controls the particle size. In some embodiments, the terminal sterilization of the dry powder extract occurs by a process such as γ sterilization. In some embodiments, the fetal support tissue can be frozen and crushed to form a fine powder extract or liquefied. In some embodiments, the frozen and crushed fetal support tissue as a powder extract can be further sieved to form particles of different sizes. In some embodiments, the frozen and crushed fetal support tissue as a fine powder can be terminally sterilized, for example, using γ sterilization. In some embodiments, the fetal support tissue extract can be liquefied and can be filtered, centrifuged or a combination thereof to sterilize it (filter pore size is equal to or less than about 0.2um) or control particle size. In some embodiments, the fetal support tissue can undergo decellularization, inactivation, micronization, frozen and crushed or a combination thereof. In some embodiments, the micronized, frozen and crushed or decellularized, inactivated fetal support tissue extract can be further filtered, centrifuged (liquid form) or sieved (dry powder) in various ways (or a combination thereof) to produce a fetal support tissue product with different particle sizes. In some embodiments, the fetal support tissue may not be mixed prior to processing (by micronization, cryo-pulverization, decellularization, inactivation, centrifugation, sterilization such as gamma sterilization, size filtration, or a combination thereof) to produce a fetal support tissue product having different particle sizes.

[0104] In some embodiments, the fetal support tissue can be freeze-dried and processed by micronization, cryo-crushing, decellularization or inactivation or a combination thereof before mixing. For example, the fetal support tissue can be freeze-dried to micronization, cryo-crushing, decellularization or inactivation or a combination thereof, further sieved (dry powder) or centrifuged, filtered (liquid form) terminally sterilized (e.g., gamma irradiated) or a combination thereof to produce fetal support tissue products with different particle sizes before mixing. In some embodiments, the fetal support tissue can be freeze-dried and mixed before being processed by micronization, cryo-crushing, decellularization, inactivation or a combination thereof. In some embodiments, the fetal support tissue can be freeze-dried and mixed before being micronized, cryo-crushed, decellularized, inactivation or a combination thereof, and can undergo sieving (dry powder) and various terminal sterilization procedures, or if it is in liquid form, it can be centrifuged, filtered or a combination thereof to produce a filtered sterilized fetal support tissue product or a fetal support tissue product of different particle sizes.

[0105] In some embodiments, the mixed fetal support tissue may be freeze-dried prior to processing by micronization, cryo-crushing, decellularization, inactivation, or a combination thereof, and may be freeze-dried prior to sieving and / or sterilizing a dry powder fetal support tissue extract or centrifuging, sterilizing, filtering a liquid form of a fetal support tissue extract / product, or a combination thereof, to produce fetal support tissue products of varying particle sizes. In some embodiments, the mixed fetal support tissue may not be freeze-dried prior to processing by micronization, cryo-crushing, decellularization, inactivation, or a combination thereof. In some embodiments, the fetal support tissue may not be freeze-dried prior to sieving and / or sterilizing a dry powder fetal support tissue extract or centrifuging, filtering a liquid form of a fetal support tissue extract / product, or a combination thereof, to produce fetal support tissue products of varying particle sizes.

[0106] In some embodiments, the particle size of the treated mixed fetal support tissue product may be less than 5 μm. In some embodiments, the particle size of the mixed treated fetal support tissue product may be less than about 5 μm. In some embodiments, the particle size of the mixed treated fetal support tissue may be from about 5 μm to about 10 μm. In some embodiments, the particle size of the mixed treated fetal support tissue product may be less than 1 μm, about less than 1 μm, about less than 10 μm to about 15 μm. In some embodiments, the mixed fetal support tissue has a particle size of any range disclosed in the specification and claims as disclosed herein, and any combination thereof.

[0107] In some embodiments, the particle size of the fetal support tissue product before mixing may be less than 5 μm. In some embodiments, the particle size of the treated fetal support tissue product before mixing may be less than about 5 μm. In some embodiments, the particle size of the treated fetal support tissue product before mixing may be from about 5 μm to about 10 μm. In some embodiments, the particle size of the treated fetal support tissue product before mixing may be less than 1 μm, about less than 1 μm, about less than 10 μm to about 15 μm. In some embodiments, the fetal support tissue before mixing has a particle size of any range disclosed in the specification and claims of the present disclosure, and any combination thereof. In some embodiments, after mixing the fetal support tissue, the fetal support tissue has a particle size of any range disclosed in the specification and claims of the present disclosure, and any combination thereof. In some embodiments, the particle size of the mixed fetal support tissue product may be less than 5 μm. In some embodiments, the particle size of the mixed treated fetal support tissue product may be less than about 5 μm. In some embodiments, the particle size of the mixed treated fetal support tissue may be from about 5 μm to about 10 μm. In some embodiments, the particle size of the mixed processed fetal support tissue product can be less than 1 μm, about less than 1 μm, about less than 10 μm to about 15 μm. In some embodiments, the mixed fetal support tissue has a particle size in any range as disclosed in the specification and claims, and any combination thereof.

[0108] In some cases, the mixing step includes a sieving step. In some embodiments, the sieving step controls the maximum particle size of the mixed composition (e.g., bulk drug substance). In some embodiments, the average pore size of the sieve is about 0.1-0.2 μm or less, 0.2-0.3 μm or less, 0.3-0.4 μm or less, 0.4-0.5 μm or less, 0.5-0.6 μm or less, 0.6-0.7 μm or less, 0.7-0.8 μm or less, 0.8-0.9 μm or less, 0.9-1 μm or less, 1-2 μm or less, 2- 3 μm or less, 3-4 μm or less, 4-5 μm or less, 5-10 μm or less, 10-20 μm or less, 20-30 μm or less, 30-40 μm or less, 40-50 μm or less, 50-100 μm or less, 100-150 μm or less, 150-200 μm or less, 200-250 μm or less, or 250-300 μm or less. In some cases, the mixing step produces a composition having high yield of HC-HA / PTX3, HA and other target proteins, improved stability, reduced variability, improved potency, uniformity, consistency, or a combination thereof, compared to a composition produced by the same method but without the mixing step.

[0109] In some embodiments, when the fetal support tissue extract is administered by inhalation or intravenously as disclosed herein, the particle size is strictly adhered to. Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims).

[0110] Preparation of mixed compositions

[0111] In some aspects, the blended compositions disclosed herein can be prepared by blending fetal support tissues from multiple donors and compared to a non-blended composition (referred to as FLO). Prior to blending, each individual blended micronized drug substance can be analyzed for hyaluronic acid content, total protein, and ODI-TRAP. Figure 1A-1B Exemplary results from such a procedure are shown in , where the results are combined to give a weighted average of each result before mixing, where a mixing process from multiple donors is described. In some embodiments, a composition made by mixing of donor fetal support tissue samples can be used for stability studies. As disclosed herein, the results of this procedure meet the acceptance criteria of T=0, 1, 3, and 6 months. The data range for each CQA is shown in Tables 1 and 2.

[0112] Extraction and centrifugation

[0113] In some embodiments, the extraction step produces a mixed fetal support tissue product in dry powder or liquid form. In some embodiments, the liquid form is centrifuged and purified to control particle size, for example, to control the particle size of fetal support tissue containing HC-HA / PTX3, HA, growth factors, cytokines and other bioactive components. In some embodiments, extracts, liquids, or powders of the aforementioned fetal support tissues may be used to purify nHC-HA / PTX3 complexes by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration or differential solubility, ethanol precipitation, or by any other available protein purification technique (see, e.g., Scopes, Protein Purification Principles and Practice 2nd ed., Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (Eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (Eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, all incorporated herein by reference).

[0114] In some embodiments, nHC-HA / PTX3 is isolated from an extract of mixed fetal support tissue. In some embodiments, the extract is prepared from an amniotic membrane extract. In some embodiments, the extract is prepared from an umbilical cord extract. In some embodiments, the umbilical cord extract includes umbilical cord matrix and / or Wharton's jelly or a combination thereof. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation using a CsCl / 4-6M guanidine hydrochloride gradient. In some embodiments, the extract is prepared by at least 2 rounds of ultracentrifugation. In some embodiments, the extract is prepared by more than 2 rounds of ultracentrifugation (ie, nHC-HA / PTX3 2). In some embodiments, the extract is prepared by at least 4 rounds of ultracentrifugation (ie, nHC-HA / PTX3 4). In some embodiments, the nHC-HA / PTX3 complex contains small leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 complex comprises HC1, HA, PTX3, and / or small leucine-rich proteoglycans.

[0115] In some embodiments, the extraction is performed on fetal support tissue. In some embodiments, the extraction includes separating the target protein from other components of the fetal support tissue. In some embodiments, the extraction is performed on frozen and pulverized fetal support tissue. In some embodiments, the extraction includes separating the target protein from other components of frozen and pulverized fetal support tissue. In some embodiments, such extraction with or without centrifugation is intended to preserve or retain HA, which is also present in nHC-HA / PTX3.

[0116] In some embodiments, the extract is prepared by extraction in an excipient. In some embodiments, the excipient is saline, water, structured water, water for injection (WFI), or a combination thereof. In some embodiments, the excipient is WFI. In some embodiments, using WFI as an excipient can produce a higher recovery of HA and total protein compared to saline or structured water. In some embodiments, the extraction extracts a majority of the nHC-HA / PTX3 in saline or WFI.

[0117] In some embodiments, the extraction is carried out for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours or about 48 hours to 72 hours. In some embodiments, the extraction is carried out for about 1 hour. In some embodiments, the extraction is carried out in WFI for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours or about 48 hours to 72 hours. In some embodiments, the extraction is carried out in WFI for about 1 hour. In some embodiments, the extraction is performed in saline for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours. In some embodiments, the extraction is performed in saline for about 1 hour.

[0118] In some embodiments, the extraction is carried out at a temperature of about 4°C. In some embodiments, the extraction is carried out at a temperature of about 3°C, 4°C, 5°C or 6°C. In some embodiments, the extraction is carried out in WFI at a temperature of about 3°C, 4°C, 5°C or 6°C. In some embodiments, the extraction is carried out in WFI at a temperature of about 4°C. In some embodiments, the extraction is carried out in WFI at a temperature of about 4°C for about 1 hour. In some embodiments, the extraction is carried out in WFI at a temperature of about 4°C for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours. In some embodiments, the extraction is carried out in saline at a temperature of about 3°C, 4°C, 5°C or 6°C. In some embodiments, the extraction is carried out in saline at a temperature of about 4°C. In some embodiments, the extraction is performed in saline at a temperature of about 4° C. for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours. In some embodiments, the extraction is performed in saline at a temperature of about 4° C. for about 1 hour.

[0119] In some embodiments, the ratio of fetal support tissue to extraction excipient is about 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.75, 1:0.5, or 1:0.25 (weight: volume). In some embodiments, the ratio of fetal support tissue to extraction excipient is about 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.75:1, 0.5:1, or 0.25:1 (weight: volume). In some embodiments, the ratio of fetal support tissue to extraction excipient is about 1:4. In some embodiments, the extraction is performed in WFI or saline. In some embodiments, the extraction is performed in WFI. In some embodiments, the extraction is performed for about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours. In some embodiments, the extraction is performed for about 1 hour. In some embodiments, the extraction is performed at a temperature of about 3°C, 4°C, 5°C, or 6°C. In some embodiments, the extraction is performed at a temperature of about 4°C. In some embodiments, the extraction is performed in WFI at a temperature of about 4°C for about 1 hour, and the ratio of fetal support tissue to excipient is about 1:4. In some embodiments, the extraction is performed in saline at a temperature of about 4°C for about 1 hour, and the ratio of fetal support tissue to excipient is about 1:4.

[0120] In some embodiments, extraction is performed using a tube rotator. In some embodiments, the tube rotator rotates at a speed range of about 5-10rpm, 10-20rpm, 20-30rpm, 30-40rpm or 40-50rpm. In some embodiments, the tube rotator rotates at a speed of about 20rpm. In some embodiments, the tube rotator rotates about 0 to 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 12 hours, about 12 hours to 24 hours, about 24 hours to 48 hours or about 48 hours to 72 hours. In some embodiments, the tube rotator rotates for about 1 hour. In some embodiments, the tube rotator rotates at a temperature of about 3°C, 4°C, 5°C or 6°C. In some embodiments, the tube rotator rotates at a temperature of about 4°C. In some embodiments, the excipient used is saline or WFI. In some embodiments, the excipient used is WFI. In some embodiments, the ratio of fetal support tissue to extraction excipient is about 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.75, 1:0.5, or 1:0.25 (weight: volume). In some embodiments, the ratio of fetal support tissue to extraction excipient is about 1:4. In some embodiments, the ratio of fetal support tissue to extraction excipient is about 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.75:1, 0.5:1, or 0.25:1 (weight: volume). In some embodiments, the extraction is performed in WFI at a temperature of about 4°C using a tube rotator at a speed of about 20 rpm for about 1 hour. In some embodiments, the extraction is performed in saline at a temperature of about 4°C using a tube rotator at a speed of about 20 rpm for about 1 hour.

[0121] In some embodiments, the extract produced by the extraction step is centrifuged. In some embodiments, the extract is centrifuged for about 5 to 10 minutes, about 10 to 15 minutes, about 15 to 20 minutes, about 20 to 30 minutes, about 30 minutes to 1 hour or about 1 to 2 hours. In some embodiments, the extract is centrifuged for about 30 minutes. In some embodiments, the extract is centrifuged at a speed of about 3,200rcf to 10,000rcf, about 10,000rcf to 14,000rcf, about 14,000rcf to 32,000rcf or about 32,000rcf to about 48,000rcf. In some embodiments, the extract is centrifuged at a speed of about 14,000rcf or higher. In some embodiments, the extract is centrifuged at a speed of about 14,000rcf. In some embodiments, the extract is centrifuged for about 30 minutes at a speed of about 14,000rcf. In some embodiments, centrifugation does not affect or rarely affects the content of target protein in the extract.

[0122] In some embodiments, the extraction produces an extract comprising hyaluronic acid polysaccharides, which are present in nHC-HA / PTX3. In some embodiments, extraction in saline or WFI causes less damage to the nHC-HA / PTX3 complex than extraction using different excipients (e.g., structured water). In some embodiments, extraction in saline or WFI produces more HA or nHC-HA / PTX3 complex yields than extraction using different excipients (e.g., structured water). In some embodiments, the extract contains greater than about 900 μg / g HA in the extract (wet). In some embodiments, the extract contains greater than about 1000 μg / g HA in the extract (wet). In some embodiments, the extract contains greater than about 1100 μg / g HA in the extract (wet). In some embodiments, the extract contains greater than about 1200 μg / g HA in the extract (wet). Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue (including components of the fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines, or other active components disclosed in the specification and claims).

[0123] dilution

[0124] In certain embodiments, described herein is a method for processing fetal support tissue, wherein the method includes a dilution step to produce different doses of fetal support tissue products. In some embodiments, the dilution step is performed on the fetal support tissue after extraction and centrifugation steps. In some embodiments, the dilution step is performed on the fetal support tissue after extraction and centrifugation steps and before it is aseptically filtered. In some embodiments, dilution of the extract (e.g., reducing the concentration of the target protein in the extract) is achieved by mixing the extract with an excipient. In some embodiments, the excipient is saline, water, structured water, water for injection (WFI) or a combination thereof. In some embodiments, the excipient is WFI. In some embodiments, the extract is mixed with an excipient with a dilution factor of about 1 to 1.5, about 1.5 to 2, about 2 to 2.5, about 2.5 to 3, about 3-3.5 about 3.5 to 4, about 4 to 5, about 5 to 6, about 6 to 7, about 7 to 8, about 8 to 9 or about 9 to 10. In some embodiments, the dilution factor is greater than 5. In some embodiments, the dilution factor is greater than 10. In some embodiments, the dilution factor is about 2. In some embodiments, the excipient is WFI and the dilution factor is 2. In some embodiments, the excipient is saline and the dilution factor is 2. In some embodiments, the methods described herein further comprise mixing the extract with the excipient for about 10 to 30 minutes, about 30 minutes to 1 hour, or about 1 hour to 2 hours. In some embodiments, the extract and the excipient are mixed for about 30 minutes. In some embodiments, the extract and the excipient are mixed at a speed of about 5-10rpm, 10-20rpm, 20-30rpm, 30-40rpm, or 40-50rpm. In some embodiments, the extract and the excipient are mixed at a speed of about 20rpm. In some embodiments, the extract and the excipient are mixed at a speed of about 20rpm for about 1 hour. In some embodiments, the extract and the excipient are mixed at a temperature of about 4°C. In some embodiments, the extract and the excipient are mixed at a speed of about 20rpm at a temperature of about 4°C for about 1 hour. In some embodiments, the extract is diluted before the filtering step. In some embodiments, the extract is diluted after the filtering step.

[0125] In some embodiments, dilution increases the filtration rate or the recovery rate of one or more target proteins after filtration, the recovery rate of one or more target proteins, or the effectiveness of the extract (e.g., measured by ODI-TRAP assay, M2 assay, NO assay and / or WST-1 assay) or a combination thereof, compared to an undiluted extract comprising the same fetal support tissue and excipients. In some embodiments, the diluted fetal support tissue comprises about 1 μg / ml to about 150 μg / ml of hyaluronic acid polysaccharide (HA). In some embodiments, the diluted fetal support tissue comprises about 1 μg / ml to about 90 μg / ml of hyaluronic acid polysaccharide (HA). In some embodiments, the diluted fetal support tissue comprises about 90 μg / ml to about 150 μg / ml of hyaluronic acid polysaccharide (HA).

[0126] Validation assay : In some embodiments, the methods disclosed herein are used to validate assays for mixed fetal support tissue products. In some embodiments, an assay for validating a mixed fetal support tissue product, extract, composition, or combination thereof is an assay or method comprising performing a validation assay on a mixed fetal support tissue, wherein the validation assay determines the amount and / or activity of a native HC-HA / PTX3 complex. In some embodiments, the assay disclosed herein validates the concentration range, potency, identity, preservation, presence of nHC-HA / PTX3, proteoglycans, cytokines, growth factors, and other biological components in fetal support tissue. In some embodiments, it is a method or assay for determining the degradation of the nHC-HA / PTX3 complex or any other component of a mixed fetal support tissue. In some embodiments, for example, when a degradation assay monitors the degradation of nHC-HA / PTX3 or the degradation of any component of a fetal support tissue, a method for monitoring the degradation of the nHC-HA / PTX3 complex or any other factor in a fetal support tissue can be performed. Such validation assays described herein include ODI-TRAP (e.g., Figure 3 ), NO production / release (e.g. Figure 5 ), M2-IL-12 release (e.g., Figure 4 ) or total HA content (e.g. Figure 7 ), can be used to monitor the presence, potency and / or activity of nHC-HA / PTX3 at any stage of processing, including, but not limited to, before mixing, after mixing, before freeze drying, after freeze drying, before micronization, after micronization, or after storage of finished product to monitor stability or potency. Assay Matrix: WST-1 Assay: Anticancer Potency of HC-HA / PTX3 Complex and HC-HA / PTX3 Extract

[0127] In some embodiments, when the HA content of the composition is at least 200 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 250 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 300 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 400 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 500 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 600 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 700 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 800 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 900 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay. In some embodiments, when the HA content of the composition is at least 1,000 μg / ml, the composition exhibits statistically significant inhibition of TRAP activity compared to the positive control in the ODI-TRAP assay.

[0128] In some embodiments, disclosed herein is a composition wherein at least 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, 400 μg / ml or at least 500 μg / ml of HA content significantly inhibits TRAP activity, wherein the inhibition is at least 70% inhibition of TRAP activity. In some embodiments, disclosed herein is a composition wherein the significant inhibition of TRAP activity is at least 80% inhibition of TRAP activity. In some embodiments, disclosed herein is a composition wherein the significant inhibition of TRAP activity is at least 85% inhibition of TRAP activity. In some embodiments, the HA content of the composition is at least 200 μg / ml. In some embodiments, the HA content of the composition is at least 250 μg / ml. In some embodiments, the HA content of the composition is at least 300 μg / ml. In some embodiments, the HA content of the composition is at least 350 μg / ml. In some embodiments, the HA content of the composition is about 300 μg / ml.

[0129] In some embodiments, therapeutic efficacy is determined by WST-1 assay. For example, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded between 1 μg / ml–1,500 μg / ml (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 1 μg / ml. In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 10 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 50 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 100 μg / ml or less. In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 200 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 250 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 300 μg / ml or less.In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 350 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 400 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 500 μg / ml or less. In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 550 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 600 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 650 μg / ml or less. In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 700 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 750 μg / ml or less (p<0.05).In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 800 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 850 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 900 μg / ml or less. In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 950 μg / ml or less (p<0.05). In some embodiments, a mixed fetal support tissue extract comprising HC-HA / PTX3 or a tissue extract comprising HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at least 1,000 μg / ml or less (p<0.05). In some embodiments, a mixed fetal supporting tissue extract comprising HC-HA / PTX3 or a tissue extract containing HC-HA / PTX3 (e.g., an umbilical cord extract but without HA) can inhibit cancer or inhibit tumor metabolic acidity compared to a positive control when the extract is loaded at about 90 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 500 μg / ml, or more than 1,500 μg / ml (p<0.05).

[0130] use ODI-TRAP Assay Matrix Determining the identity and potency of fetal support tissue compositions In one aspect described herein, a mixed composition of fetal support tissue comprises a potency determined by ODI-TRAP. The cell-based ODI-TRAP assay is a quantitative biological test that measures the specific activity of mixed fetal support tissue based on its activity to inhibit TRAP activity. Inhibitory activity can be assessed by comparative assays to assess and detect general inhibition of ODI-TRAP (e.g., Figure 6B), or setting up the assay to perform PCR on a single donor sample batch (e.g., FLO; Fig. 10A ) and the inhibition detected in mixed sample batches ( Fig. 10B) in a TRAP inhibition assay. In certain embodiments, the mixed fetal support tissue possesses sufficient potency if it exhibits a statistically significant difference in the TRAP assay compared to the positive control for fetal support tissue having an HA content of at least 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, 400 μg / ml, 450 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1,000 μg / ml. In certain embodiments, for fetal support tissues with a HA content of at least 5 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with a HA content of at least 10 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with a HA content of at least 15 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with a HA content of at least 20 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with a HA content of at least 30 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with a HA content of at least 40 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 50 μg / ml, a mixed fetal support tissue has sufficient efficacy if a statistically significant difference is shown compared to a positive control. In certain embodiments, for fetal support tissues having an HA content of at least 100 μg / ml, a mixed fetal support tissue has sufficient efficacy if a statistically significant difference is shown compared to a positive control. In certain embodiments, for fetal support tissues having an HA content of at least 150 μg / ml, a mixed fetal support tissue has sufficient efficacy if a statistically significant difference is shown compared to a positive control.In certain embodiments, for fetal support tissues with an HA content of at least 200 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with an HA content of at least 250 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with an HA content of at least 300 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with an HA content of at least 350 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues with an HA content of at least 400 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 500 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 600 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 700 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 800 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, for fetal support tissues having an HA content of at least 900 μg / ml, if a statistically significant difference is shown compared to a positive control, the mixed fetal support tissue has sufficient efficacy. In certain embodiments, the mixed fetal support tissue has sufficient potency if it exhibits a statistically significant difference compared to a positive control for a fetal support tissue having an HA content of at least 1,000 μg / ml.

[0131] In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 60% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 65% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 70% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 75% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 80% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 85% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 90% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 95% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 97% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 100 μg / ml of HA exerts at least 98% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP potency if at least about 100 μg / ml of HA exerts at least 99% inhibition of TRAP.

[0132] In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 60% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 65% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 70% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 75% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 80% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 85% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 90% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 95% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 97% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts at least 98% of the TRAP inhibitory effect. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP potency if at least about 300 μg / ml of HA exerts at least 99% inhibition of TRAP.

[0133] In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 60% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 65% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 70% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 75% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 80% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 85% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 90% TRAP inhibition. In some embodiments, the mixed fetal support tissue exhibits sufficient TRAP efficacy if at least about 300 μg / ml of HA exerts greater than 95% TRAP inhibition.

[0134] HA assay:Hyaluronic acid (HA) is the main extracellular matrix in AM and UC, and is a component of the HC-HA / PTX3 complex. The HA assay disclosed herein has been validated and tested to evaluate the stability of fetal support tissue extracts (e.g., HC-HA / PTX3). HA assays are essential for the release of compositions or drugs of fetal support tissues, wherein HA assays are identity and efficacy measurements. In certain embodiments, if the HA content obtained is at least 90 μg / ml, the hyaluronic acid (HA) in the mixed fetal support tissue and / or product has sufficient efficacy. In some embodiments, when the HA test kit has a "not less than" (NLT) content of at least 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA efficacy. In some embodiments, when the HA test kit has at least 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, 170 μg / ml, 180 μg / ml, 190 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml 1, 400μg / ml, 450μg / ml, 500μg / ml, 550μg / ml, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 850μg / ml, 900μg / ml, 950μg / ml, 1,000μg / ml, 2,000μg / ml, 3,000μg / ml "not less than" (NLT) content, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has a "not less than" (NLT) content of at least 50μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 60μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, the mixed fetal support tissue extract and / or composition has sufficient HA potency when the HA test kit has an NLT content of at least 70 μg / ml. In some embodiments, the mixed fetal support tissue extract and / or composition has sufficient HA potency when the HA test kit has an NLT content of at least 80 μg / ml.In some embodiments, when the HA test kit has an NLT content of at least 90 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 100 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 110 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 120 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 130 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 140 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 150 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 160 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 170 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 180 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 190 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 200 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 250 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 300 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 350 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency.In some embodiments, when the HA test kit has an NLT content of at least 400 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 450 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 500 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 550 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 600 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 650 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 700 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 750 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 800 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 850 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 900 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency. In some embodiments, when the HA test kit has an NLT content of at least 1,000 μg / ml, the mixed fetal support tissue extract and / or composition has sufficient HA potency.

[0135] Bicinchoninic Acid (BCA) Protein Assay (BCA): The BCA assay is a quantitative and analytical assay used to measure the total protein content of fetal support tissues and / or products, including HC-HA / PTX3 complexes, tissue matrix, growth factors / cytokines, and other bioactive factors found in tissues or drug substances. The BCA assay is one of the initial product release tests to assess identity and potency. The BCA assay disclosed herein has been validated and tested to assess the stability of fetal support tissue extracts (e.g., HC-HA / PTX3). The BCA assay is critical for the release of compositions or drugs from fetal support tissues where the BCA assay is an identity and potency measurement. In certain embodiments, the total protein content is "not less than" (NLT) of at least 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, 170 μg / ml, 180 μg / ml, 190 μg / ml, 200 μg / ml, 220 μg / ml, 230 μg / ml, 240 μg / ml, 250 μg / ml, 260 μg / ml, 270 μg / ml, 280 μg / ml, 290 μg / ml, 300 μg / ml, 310 μg / ml, 320 μg / ml, 330 μg / ml, 340 μg / ml, 350 μg / ml, 360 μg / ml, 370 μg / ml, 380 μg / ml, 390 μg / ml, 400 μg / ml, 410 μg / ml, 420 μg / ml, 430 μg / ml, 440 μg / ml The total protein content in the mixed fetal support tissue and / or product is sufficient to be effective if the total protein content in the mixed fetal support tissue and / or product is 10μg / ml, 320μg / ml, 330μg / ml, 340μg / ml, 350μg / ml, 360μg / ml, 370μg / ml, 380μg / ml, 390μg / ml, 400μg / ml, 450μg / ml, 500μg / ml, 550μg / ml, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 850μg / ml, 900μg / ml, 950μg / ml, 970μg / ml, 1,000μg / ml, 2,000μg / ml, 3,000μg / ml, 4,000μg / ml, 5,000μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient potency if the total protein content of NLT detected by the BCA assay is at least 30 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient potency if the total protein content of NLT detected by the BCA assay is at least 40 μg / ml.In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 50 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 60 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 70 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 80 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 90 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 100 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 110 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 120 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 130 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 140 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 150 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 160 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 170 μg / ml.In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 180 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 190 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 200 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 210 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 220 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 230 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 240 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 250 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 260 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 270 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 280 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 290 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 300 μg / ml.In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 310 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 320 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 330 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 340 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 350 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 360 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 370 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 380 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 390 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 400 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 450 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 500 μg / ml. In certain embodiments, the total protein content of the mixed fetal support tissue and / or product has sufficient efficacy if the total protein content of NLT detected by the BCA assay is at least 550 μg / ml.In certain embodiments, if the BCA assay detects a total protein content of NLT of at least 600 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the BCA assay detects a total protein content of NLT of at least 650 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the BCA assay detects a total protein content of NLT of at least 700 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the BCA assay detects a total protein content of NLT of at least 750 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the BCA assay detects a total protein content of NLT of at least 800 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 850 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 900 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 950 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 970 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy. In certain embodiments, if the total protein content of NLT detected by the BCA assay is at least 1,000 μg / ml, the total protein content in the mixed fetal support tissue and / or product has sufficient efficacy.

[0136] M2 polarization assay based on NO measurement: Lipopolysaccharide (LPS) and interferon gamma (IFNγ) can stimulate the mouse macrophage cell line RAW264.7 cells to polarize into M1 macrophages (pro-inflammatory phenotype) and produce high nitric oxide (NO) by upregulating inducible NO synthase (iNOS) (pro-inflammatory mediator), while M2 macrophages (anti-inflammatory phenotype) produce low NO. Therefore, inhibition of NO production indicates anti-inflammatory efficacy by downregulating M1 macrophages. Disclosed herein in some embodiments, the NO assay can demonstrate the anti-inflammatory effect of mixed fetal support tissue HC-HA / PTX3 or tissue extracts containing HC-HA / PTX3 (e.g., Figure 5In some embodiments, the mixed fetal support tissue has sufficient efficacy if the fetal support tissue exhibits a statistically significant difference compared to the positive control. Filtration of liquid extracts: sterilization and particle separation

[0137] In some embodiments, the fetal support tissue is sterilized by any suitable method. In some embodiments, the fetal support tissue is sterilized by irradiation, such as, for example, by gamma irradiation, by exposure to a chemical sterilant, by heating, by filtration, by exposure to ethylene oxide gas, by filtration through a membrane with an appropriate pore size, by electron beam (E-beam) irradiation, or by any treatment that renders the fetal support tissue free of contamination by viable microorganisms while preserving the biological activity of the bioactive components disclosed herein.

[0138] In some embodiments, different filters with different pore sizes can be used for filtering to control particle size as a purpose or to sterilize liquid tissue extracts. In some embodiments, fetal support tissue is sterilized by filtration. In some embodiments, sterilizing fetal support tissue by filtration includes passing fetal support tissue through a filter. In some embodiments, the selection of filter pore size is to prevent bacteria, yeast, mold or virus from passing through the filter. In some cases where filtration sterilization is used to sterilize liquid forms, the filtration process uses a filter with a pore size less than or equal to 0.2um.

[0139] In some embodiments, the fetal support tissue extract is filtered to control particle size. In some embodiments, the filter comprises pores having an average size of about 0.1-0.2 μm or less, 0.2-0.3 μm or less, 0.3-0.4 μm or less, 0.4-0.5 μm or less, 0.5-0.6 μm or less, 0.6-0.7 μm or less, 0.7-0.8 μm or less, 0.8-0.9 μm or less, 0.9-1 μm or less, 1-2 μm or less, 2-3 μm or less, 3-4 μm or less, 4-5 μm or less, 5-10 μm or less, 10-20 μm or less, 20-30 μm or less, 30-40 μm or less, 40-50 μm or less, 50-100 μm or less. In some embodiments, the filter has an average pore size of about .05–0.2 μm. In some embodiments, the filter comprises pores having an average size of about 0.4 μm or less. In some embodiments, the filter comprises pores having an average size of about 0.3 μm or less. In some embodiments, the filter comprises pores having an average size of about 0.2 μm or less. In some embodiments, the filter comprises pores having an average size of about 0.2 μm. In some embodiments, sterilizing by filtration comprises passing the fetal support tissue through a first filter and a second filter. In some embodiments, the first filter comprises an average pore size that is greater than the average pore size of the second filter. In some embodiments, the first filter or the second filter has an average pore size of about 0.1-0.2 μm or less, 0.2-0.3 μm or less, 0.3-0.4 μm or less, 0.4-0.5 μm or less, 0.5-0.6 μm or less, 0.6-0.7 μm or less, 0.7-0.8 μm or less, 0.8-0.9 μm or less, 0.9-1 μm or less, 1-2 μm or less, 2-3 μm or less, 3-4 μm or less, 4-5 μm or less, 5-10 μm or less. In some embodiments, each of the first filter and the second filter has an average pore size of about 0.05-0.2 μm. In some embodiments, the first filter has an average pore size of about 0.6 μm or less, and the second filter has an average pore size of about 0.4 μm or less. In some embodiments, the first filter has an average pore size of about 0.5 μm or less, and the second filter has an average pore size of about 0.3 μm or less. In some embodiments, the first filter has an average pore size of about 0.45 μm or less, and the second filter has an average pore size of about 0.2 μm or less. In some embodiments, the first filter has an average pore size of about 0.45 μm and the second filter has an average pore size of about 0.2 μm.

[0140] In some embodiments, any filter described herein is contained in a sterilization unit. In some embodiments, any filter described herein is a membrane comprising polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene, polyethylene, polyamide, cellulose, nitrocellulose, nylon or a combination thereof. In some embodiments, any filter described herein has been sterilized by gamma irradiation. In some embodiments, the filtration pressure during filtration is about 0-10psi, about 10-20psi, about 20-30psi, about 30-40psi, about 40-50psi, about 50-60psi, about 60-70psi, about 70-80psi, about 80-90psi or about 90-100psi. In some embodiments, the effective filtration area of ​​any filter described herein is about 0-20cm 2 , about 20-40cm 2 、About 40-60cm 2 、About 60-80cm 2 、About 80-100cm 2 , about 100-120cm 2 , about 120-140cm 2 , about 140-160cm 2 , about 160-180cm 2 or about 180-200cm 2In some embodiments, the overall diameter of the filter is about 0-10 mm, about 20-20 mm, about 20-30 mm, about 30-40 mm, about 40-50 mm, about 50-60 mm, about 60-70 mm, about 70-80 mm, about 80-90 mm, about 90-100 mm, about 100-200 mm, about 200-300 mm, about 300-400 mm, about 400-500 mm, about 500-600 mm, about 600-700 mm, about 700-800 mm, about 800-900 mm, or about 900-1000 mm. In some embodiments, the overall diameter of the filter is about 67 mm. In some embodiments, the overall diameter of the filter is about 68 mm. In some embodiments, the overall height of the filter is about 0-10mm, about 20-20mm, about 20-30mm, about 30-40mm, about 40-50mm, about 50-60mm, about 60-70mm, about 70-80mm, about 80-90mm, about 90-100mm, about 100-200mm, about 200-300mm, about 300-400mm, about 400-500mm, about 500-600mm, about 600-700mm, about 700-800mm, about 800-900mm or about 900-1000mm. In some embodiments, the overall height of the filter is about 82mm. In some embodiments, the overall height of the filter is about 83mm. In some embodiments, the filter has been successfully tested by manufacturing forward flow. In some embodiments, the filter has a forward flow rate limit of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or greater than 1.0 mL / minute. In some embodiments, when fully wetted with water, the filter has a forward flow rate limit of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or greater than 1.0 mL / minute at a test pressure of about 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900 or greater than 2900 mbar. In some embodiments, when fully wetted with water, the filter has a forward flow limit of about 0.58 mL / minute at a test pressure of about 2760 mbar. In some embodiments, when fully wetted with water, the filter has a forward flow limit of about 0.40-0.50 mL / min, 0.50-0.60 mL / min, or 0.60-0.70 mL / min at a test pressure of about 2700-2800 mbar. In some embodiments, the forward flow test limit has been verified to remove bacteria by correlating the forward flow limit with a microbial challenge test.In some embodiments, fetal support tissue is tested for retention of acceptable challenge microorganisms using procedures consistent with applicable Food and Drug Administration guidelines to verify bacterial retention of the filter.

[0141] In some embodiments, the fetal support tissue powder products disclosed herein are terminally sterilized by any suitable (e.g., medically acceptable) method. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to gamma radiation for a period of time sufficient to sterilize the fetal support tissue powder products disclosed herein.

[0142] In some embodiments, the fetal support tissue powder products disclosed herein are exposed to about 10 to about 75 kilograys (kGy) of gamma radiation for a period of time sufficient to sterilize the fetal support tissue powder products. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to about 10 to about 30kGy of gamma radiation for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to about 15 to about 30kGy of gamma radiation for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to about 25kGy of gamma radiation for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to about 17.5kGy of gamma radiation for a period of time sufficient to sterilize the fetal support tissue powder products.

[0143] In some embodiments, the fetal support tissue powder product disclosed herein is sterilized by electron beam (E-beam). In some embodiments, the fetal support tissue disclosed herein is exposed to E-beam radiation of about 10 to about 75 kilograys, for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue disclosed herein is exposed to E-beam radiation of about 10 to about 30kGy, for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue disclosed herein is exposed to E-beam radiation of about 15 to about 30kGy, for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue disclosed herein is exposed to E-beam radiation of about 25kGy, for a period of time sufficient to sterilize the fetal support tissue. In some embodiments, the fetal support tissue disclosed herein is exposed to E-beam radiation of about 17.5kGy, for a period of time sufficient to sterilize the fetal support tissue.

[0144] In some embodiments, the fetal support tissue powder products disclosed herein are exposed to an electron beam for a period of time sufficient to sterilize the fetal support tissue powder product. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to X-ray radiation for a period of time sufficient to sterilize the fetal support tissue powder product. In some embodiments, the fetal support tissue powder products disclosed herein are exposed to UV radiation for a period of time sufficient to sterilize the fetal support tissue powder product.

[0145] In certain embodiments, provided herein are methods for preparing fetal support tissues, wherein the methods result in an increase in the percentage of recovered HA. In some embodiments, at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% HA is recovered. In some embodiments, at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% HA is recovered. In some embodiments, at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% HA is recovered. In some embodiments, at least or about 50% HA is recovered. In some embodiments, at least or about 55% HA is recovered. In some embodiments, at least or about 60% HA is recovered. In some embodiments, at least or about 65% of the HA is recovered. In some embodiments, at least or about 70% of the HA is recovered. In some embodiments, at least or about 75% of the HA is recovered. In some embodiments, at least or about 80% of the HA is recovered. In some embodiments, at least or about 85% of the HA is recovered. In some embodiments, at least or about 85% of the HA is recovered. In some embodiments, at least or about 95% of the HA is recovered. In some embodiments, at least or about 99% of the HA is recovered. In some embodiments, at least or about 99% or more of the HA is recovered. In some embodiments, the HA is HMW HA. In some embodiments, the method results in the preservation of nHC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by the recovery of at least or about 75%, 80%, 85%, 90%, 95%, 99%, or more than 99% of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 75% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 80% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 85% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 90% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 95% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 99% recovery of HC-HA / PTX3. In some embodiments, the potency of nHC-HA / PTX3 is preserved by at least or about 99% or more recovery of HC-HA / PTX3.

[0146] In certain embodiments, the methods described herein result in the removal of particulate matter or degradants. In some embodiments, the methods described herein result in the removal of at least or about 75%, 80%, 85%, 90%, 95%, 99%, or more than 99% of particulate matter or degradants. In some embodiments, the particulate matter or degradants include chlorides.

[0147] Rehydration

[0148] In some embodiments, the fetal support tissue is partially or completely rehydrated so that the powder extract becomes a liquid extract. In some embodiments, rehydration allows the fetal support tissue as a dry powder to be reconstituted into a liquid form using water or a suitable buffer. In some embodiments, the rehydrated fetal support tissue or tissue extract is further filtered for sterilization purposes. Sterilization of the rehydrated fetal support tissue extract is accomplished by filtering the extract using a filter having a pore size of about less than or equal to 0.2 μm.

[0149] In some embodiments, the rehydrated tissue extract is further filtered to control the particle size, for example, the extract is further centrifuged in various ways to produce different particle sizes for administration or preparation of fetal support tissue extract / composition. In some embodiments, the fetal support tissue is rehydrated by contacting the fetal support tissue with a buffer or with water. In some embodiments, the fetal support tissue is contacted with an isotonic buffer. In some embodiments, the fetal support tissue is contacted with saline. In some embodiments, the fetal support tissue is contacted with PBS. In some embodiments, the fetal support tissue is contacted with Ringer's solution. In some embodiments, Ringer's solution is lactate Ringer's Saline. In some embodiments, the fetal support tissue is contacted with Hartmann's solution. In some embodiments, the fetal support tissue is contacted with TRIS buffered saline. In some embodiments, the fetal support tissue is contacted with HEPES buffered saline; 50% DMEM + 50% glycerol; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% glycerol; and / or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% propylene glycol.

[0150] In some embodiments, the fetal support tissue is contacted with the buffer for 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours or more than 24 hours. In some embodiments, the UC product is contacted with the buffer for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more than 4 weeks. Disclosed herein is a process for generating fetal support tissue while preserving the biological activity of fetal support tissue (including components of fetal support tissue, such as HC-HA / PTX3, HA, growth factors, cytokines or other active components disclosed in the specification and claims). In some embodiments, the fetal support tissue is stored for later use. In some embodiments, storing the fetal support tissue will not destroy the integrity of the fetal support tissue extracellular matrix. In some embodiments, the fetal support tissue is freeze-dried. In some embodiments, the fetal support tissue is stored in any suitable storage medium.

[0151] In some embodiments, the fetal support tissue is optionally in contact with a substrate (i.e., a supporting backing). In some embodiments, the fetal support tissue is not in contact with the substrate. In some embodiments, the fetal support tissue is oriented so that the fetal support tissue is in contact with the substrate. In some embodiments, the fetal support tissue is oriented so that the matrix is ​​in contact with the substrate. In some embodiments, the fetal support tissue is oriented so that the epithelial side is in contact with the substrate.

[0152] In some embodiments, the fetal support tissue is attached to a substrate. In some embodiments, the substrate is nitrocellulose paper (NC). In some embodiments, the substrate is a nylon membrane (NM). In some embodiments, the substrate is a polyethersulfone membrane (PES).

[0153] Natural HC-HA / PTX3 (nHC-HA / PTX3) Composition

[0154] In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from amniotic tissue. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from amniotic membrane or umbilical cord. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from fresh, frozen or previously frozen placental amniotic membrane (PAM), fresh, frozen or previously frozen umbilical cord amniotic membrane (UCAM), fresh, frozen or previously frozen placenta, fresh, frozen or previously frozen umbilical cord, fresh, frozen or previously frozen chorion, fresh, frozen or previously frozen amniotic-chorionic membrane, or any combination thereof. Such tissues may be obtained from any mammal, such as, but not limited to, humans, non-human primates, cattle, or pigs, or combinations thereof.

[0155] In some embodiments, nHC-HA / PTX3 is purified by any suitable method. In some embodiments, the nHC-HA / PTX3 complex is purified by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration or differential solubility, ethanol precipitation, or by any other available protein purification technique (see, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (Eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (Eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, all incorporated herein by reference).

[0156] In some embodiments, nHC-HA / PTX3 is isolated from an extract. In some embodiments, the extract is prepared from an amniotic membrane extract. In some embodiments, the extract is prepared from an umbilical cord extract. In some embodiments, the umbilical cord extract includes umbilical cord stroma and / or Wharton's jelly. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation using a CsCl / 4-6M guanidine hydrochloride gradient.

[0157] In some embodiments, the extract is prepared by at least 2 rounds of ultracentrifugation. In some embodiments, the extract is prepared by more than 2 rounds of ultracentrifugation (i.e., nHC-HA / PTX3 round 3). In some embodiments, the extract is prepared by at least 4 rounds of ultracentrifugation (i.e., nHC-HA / PTX3 round 4). In some embodiments, the nHC-HA / PTX3 complex comprises small leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 complex comprises HCl, HA, PTX3, and / or small leucine-rich proteoglycans (SLRPs).

[0158] In some embodiments, the fetal support tissue comprises nHC-HA / PTX3 and a pharmaceutical excipient. In some embodiments, the fetal support tissue consists essentially of nHC-HA / PTX3 complexes. In some embodiments, the fetal support tissue comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In some embodiments, the appropriate formulation depends on the selected route of administration.

[0159] In some embodiments, the nHC-HA / PTX3 complex is purified by immunoaffinity chromatography, affinity chromatography, or a combination thereof. In some embodiments, anti-HC1 antibodies, anti-HC2 antibodies, or both are generated and immobilized on a solid support. In some embodiments, the HC-HA complex is bound to an antibody (e.g., by interaction of (a) anti-HC1 antibodies with HC1, (b) anti-HC2 antibodies with HC2, (c) anti-PTX antibodies with PTX3, (d) anti-SLRP antibodies with SLRP, or (e) any combination thereof). In some embodiments, HABP is generated and immobilized on a solid support.

[0160] In some embodiments, one or more antibodies are used to purify the nHC-HA / PTX3 complex from the insoluble fraction described herein. In some embodiments, an anti-SLRP antibody is used to purify the nHC-HA / PTX3 complex from the insoluble fraction described herein.

[0161] In some embodiments, the nHC-HA / PTX3 complex is purified from a soluble fraction described herein. In some embodiments, the nHC-HA / PTX3 complex is purified from a soluble fraction described herein using an anti-PTX3 antibody.

[0162] In some embodiments, fetal support tissue may include, for example, extracellular matrix, growth factors, cytokines, and components of the nHC-HA / PTX3 complex. In some embodiments, an extracellular matrix is ​​a small leucine-rich proteoglycan (SLRP). In some embodiments, the nHC-HA / PTX3 complex includes a class I, class II, or class III SLRP. In some embodiments, the small leucine-rich proteoglycan is selected from class I SLRPs, such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from class III SLRPs, such as epipycan and osteooglycin. In some embodiments, the small leucine-rich proteoglycan is selected from bikunin, decorin, biglycan, and osteonectin. In some embodiments, the small leucine-rich protein comprises glycosaminoglycans. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate.

[0163] In some embodiments, the pharmaceutical composition further includes at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further includes an adjuvant, an excipient, a preservative, a retarder, a filler, an adhesive, an adsorbent, a buffer and / or a solubilizer. Exemplary pharmaceutical compositions formulated to contain the nHC-HA / PTX3 complexes provided herein include, but are not limited to, gels, solutions, suspensions, emulsions, syrups, granules, powders, homogenates, ointments, tablets, capsules, pills, pastes, creams, lotions, patches, sticks, films, coatings, aerosols, or combinations thereof. In some embodiments, the fetal support tissue containing nHC-HA / PTX3 is a graft or sheet.

[0164] Exemplary Biological Factors in Fetal Support Tissues

[0165] Disclosed herein are fetal support tissues containing bioactive factors that can be preserved and used in various applications disclosed herein. Fetal support tissues disclosed herein, such as amniotic membranes and umbilical cords, contain a variety of innate biological factors and have a variety of important clinical effects disclosed herein. For example, nHC-HA / PTX3 complexes—high molecular weight (HMW) hyaluronic acid polysaccharides (HA) covalently linked to heavy chain (HC) 1 from inter-α-trypsin inhibitor, and further compounded with pentraxin 3 (PTX3)—as exemplary key active components of umbilical cord and amniotic membrane, responsible for wound healing effects, other extracellular matrices, growth factors and cytokines, and several other therapeutic effects (components) of fetal support tissues. Therefore, it is crucial to produce fetal support tissue products (such as amniotic membrane and umbilical cord extracts for wound healing) with high yields of HC-HA / PTX3, HA, and other target proteins. Using treatments that reduce or prevent degradation of HC-HA / PTX3 complexes and other target proteins to produce fetal support tissue products, while concentrating these biomolecules is a key factor in processing fetal support tissue compositions. Preventing degradation of HA and other target proteins is important because degradation of such proteins can render the fetal support tissue product unsuitable for use.

[0166] In certain embodiments, disclosed herein is a method for preparing a fetal support tissue product, comprising: (a) freeze drying mixed or unmixed fetal support tissue to produce micronized, cryo-pulverized, (dry or wet), inactivated, decellularized (frozen), or a combination thereof, or (b) extracting the processed fetal support tissue in an excipient to produce an extract; and (c) sterilizing the fetal support tissue by filtering the extract using a membrane having a pore size of less than about 0.2 μm. In some embodiments, the fetal support tissue is placental amniotic membrane (PAM) or substantially isolated PAM, umbilical cord amniotic membrane (UCAM) or substantially isolated UCAM, chorion or substantially isolated chorion, amnion-chorion or substantially isolated amnion-chorion, placenta or substantially isolated placenta, umbilical cord or substantially isolated umbilical cord, or any combination thereof.

[0167] In some embodiments, the diluted fetal support tissue comprises about 1 μg / mL to about 1 mg / mL of HA, some of which is present as HC-HA / PTX3.

[0168] In certain embodiments, compositions are described herein, which compositions include a therapeutically effective amount of a mixed composition including fetal support tissue. In some embodiments, the fetal support tissue includes micronized fetal support tissue. In some embodiments, the mixed composition includes fetal support tissue from multiple donors. In some embodiments, the composition includes fetal support tissue mixed from at least 15 donors. In some embodiments, the composition includes fetal support tissue mixed from at least 30 donors. In some embodiments, the composition includes fetal support tissue mixed from at least 45 donors. In some embodiments, the composition includes fetal support tissue mixed from at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 donors. In some embodiments, if the composition includes tissue from up to 5 donors, it is not mixed. In some embodiments, if the composition includes tissue from up to 9, 8, 7, 6, 5, 4, 3 or 2 donors, it is not mixed.

[0169] Mixed fetal support tissue reduces sample variability and improves sample consistency

[0170] In certain embodiments, compositions comprising mixed fetal support tissue or fetal support tissue extracts are described herein or compositions comprising reduced donor-to-donor (or inter-subject) variability compared to non-mixed (individual donor) fetal support tissue extract compositions. In some embodiments, the mixed composition has reduced variability compared to compositions comprising fetal support tissues from up to 9, 8, 7, 6, 5, 4, 3 or 2 donors. In some embodiments, the mixed composition has reduced variability compared to compositions comprising fetal support tissues from up to 5 donors. In some embodiments, the mixed composition has reduced variability compared to compositions comprising fetal support tissues from one donor. Disclosed herein are mixed fetal support tissue extracts (in any form disclosed herein) that may have reduced mixed sample-to-sample variability compared to non-mixed (individual) fetal support tissue extracts. For example, when the average percent coefficient of variation (CV%) of a mixed fetal support tissue product or extract is calculated, the mixed CV% may be at least 1 / 2, 1 / 3, 1 / 4 or more lower than the CV% of a non-mixed fetal support tissue product. Disclosed herein are mixed fetal support tissue extracts (in any form disclosed herein), including mixed products that show increased uniformity and improved sample consistency compared to the uniformity or sample consistency of non-mixed fetal support tissue extracts. For example, as determined by an ODI-TRAP assay, a BCA assay, or a HA quantitative assay, a mixed fetal support tissue extract (in any form, such as a composition, a kit, etc.) may exhibit improved consistency, wherein the mixed extract exhibits a reduced activity difference of a natural HC-HA / PTX3 complex compared to a non-mixed fetal support tissue extract (product, composition, etc.). For example, the improvement in the consistency of the mixed extract can be shown by a reduced coefficient of variation of at least 10%. In some embodiments, the reduced coefficient of variation of the mixed fetal support tissue extract can be 5% or less compared to the non-mixed fetal support tissue extract. In some embodiments, the reduced coefficient of variation of the mixed fetal support tissue extract can be 4% or less compared to the non-mixed fetal support tissue extract. In some embodiments, the reduced coefficient of variation of the mixed fetal support tissue extract can be about 3% or less compared to the non-mixed fetal support tissue extract. In some embodiments, the reduced coefficient of variation of the pooled fetal support tissue extract can be about 2% or less compared to the non-pooled fetal support tissue extract.

[0171] Nevertheless, the present disclosure retains HA molecular weight (MW) for both mixed fetal support donor samples and single donor samples and any derived mixed and non-mixed fetal support tissue extracts (compositions, kits, products, etc.); wherein mixing neither changes the HA MW nor the HC-HA bond. The mixed fetal support tissues disclosed herein and the derived extracts or products provide a higher yield of products that also have a higher uniformity, resulting in less variability between mixed samples. As described above and throughout the disclosure, the mixing of samples improves the consistency of the product while increasing protein yield (e.g., HA protein content) due to reduced variability (measured by a reduced coefficient of variation) compared to that obtained from non-mixed single donor samples and / or extracts. As disclosed herein, the present disclosure produced by the mixing process fully retains the efficacy of the fetal support tissue extract (product / composition, etc.), for example, fully retains the efficacy of the natural HC-HA / PTX3 complex, while improving the yield, uniformity and consistency of the mixed extract. Therefore, the findings of the present disclosure advocate the mixing of fetal support tissues and / or fetal support tissue products.

[0172] In some embodiments, the fetal support tissue is placental amnion, umbilical cord, umbilical cord amnion, chorion, amnion-chorion, placenta, amnion matrix, amnion glue or any combination thereof. In some embodiments, the fetal support tissue includes the extracellular matrix (ECM) of amnion (AM) and umbilical cord (UC). Fetal support tissue, such as the ECM of AM / UC contains a variety of innate biological factors, which can be used for a variety of purposes including wound healing and reducing inflammation and scar formation. Characterization data show that the extracellular matrix (ECM) of fetal support tissue, such as AM and UC, is rich in hyaluronic acid (HA) and has an important or key matrix component called HC-HA / PTX3 complex (heavy chain 1-hyaluronic acid-positive pentraxin 3). In some embodiments, the fetal support tissue includes AM and UC, wherein HC-HA / PTX3 is separated, and wherein HC-HA / PTX3 is natural (nHC-HA / PTX3). In some embodiments, the fetal support tissue includes micronized fetal support tissue.

[0173] In some embodiments, variability is determined by the average particle size in the mixed composition. In some embodiments, the mixed composition comprises no more than about 10000 particles having a diameter greater than about 10 μm. In some embodiments, the mixed composition comprises no more than about 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 250 or 100 particles having a diameter greater than about 10 μm or less than 10 μm, less than 5 μm, less than 1 μm or about 1 μm, about 1 μm to about 5 μm or about 5 μm to about 10 μm or a combination thereof. In some embodiments, the mixed composition comprises no more than about 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 250 or 100 particles having a diameter greater than about 10 μm or less than 10 μm, less than 5 μm, less than 1 μm or about 1 μm, about 1 μm to about 5 μm or about 5 μm to about 10 μm or a combination thereof. In some embodiments, the mixed composition comprises no more than about 1000 particles having a diameter greater than about 25 μm. In some embodiments, the mixed composition comprises no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 or 100 particles having a diameter greater than about 10, 15, 20, 25, 30, 35, 40, 45 or 50 μm. In some embodiments, the mixed composition comprises no more than about 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 250, or 100 particles having a diameter greater than about 10, 15, 20, 25, 30, 35, 40, 45, or 50 μm.

[0174] In certain embodiments, compositions comprising a therapeutically effective amount of a mixed composition are described herein, the mixed composition comprising fetal support tissue, wherein the fetal support tissue comprises a particle size of a specific diameter. In certain embodiments, compositions comprising a therapeutically effective amount of a mixed composition are further described herein, the mixed composition comprising fetal support tissue, wherein the fetal support tissue comprises a particle size of about 20 μm to about 240 μm in diameter. In some embodiments, the fetal support tissue comprises micronized fetal support tissue.

[0175] In some embodiments, the fetal support tissue comprises an average particle size of about 10 μm to about 300 μm, about 10 μm to about 250 μm, about 10 μm to about 200 μm, about 10 μm to about 150 μm, about 10 μm to about 100 μm, or about 10 μm to about 50 μm. In some embodiments, the fetal support tissue comprises an average particle size of about 20 μm to about 200 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 20 to about 40 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 20 μm to about 300 μm, about 20 μm to about 250 μm, about 20 μm to about 200 μm, about 20 μm to about 150 μm, about 20 μm to about 100 μm, about 20 μm to about 50 μm, about 50 μm to about 300 μm, about 50 μm to about 250 μm, about 50 μm to about 200 μm, about 50 μm to about 150 μm, or about 50 μm to about 100 μm. In some embodiments, the fetal support tissue comprises an average particle size of about 60 μm to about 120 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 140 μm to about 240 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of at least or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 100 μm in diameter.

[0176] In some embodiments, the fetal support tissue comprises an average particle size of about 0.010 μm to about 0.3 μm, about 0.01 μm to about 0.25 μm, about 0.01 μm to about 0.2 μm, about 0.01 μm to about 0.15 μm, about 0.01 μm to about 0.1 μm, about 0.01 μm to about 0.5 μm, 0.10 μm to about 0.3 μm, about 0.1 μm to about 0.25 μm, about 0.1 μm to about 0.2 μm, about 0.1 μm to about 0.15 μm, about 0.1 μm to about 0.25 μm, or about 0.1 μm to about 0.5 μm. In some embodiments, the fetal support tissue comprises an average particle size of about 0.20 μm to about 2.0 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 0.20 μm to about 4.0 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 0.20 μm to about 3.0 μm, about 0.20 μm to about 2.5 μm, about 0.20 μm to about 2.0 μm, about 0.20 μm to about 1.5 μm, about 0.20 μm to about 1.0 μm, about 0.20 μm to about 5.0 μm, about 0.50 μm to about 3.0 μm, about 0.50 μm to about 2.5 μm, about 0.50 μm to about 2.0 μm, about 0.50 μm to about 1.5 μm, or about 0.50 μm to about 1.0 μm. In some embodiments, the fetal support tissue comprises an average particle size of about 0.60 μm to about 1.2 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of at least or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.3, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 1.0, 1.5, 2.0, 2.5, 3.0, or more than 3.0 μm in diameter. In some embodiments, the fetal support tissue comprises an average particle size of about 0.50 μm in diameter. Disclosed herein are processes for generating fetal support tissue while preserving the biological activity of the fetal support tissue, including components of the fetal support tissue, such as, for example, HA (which is present as nHC-HA / PTX3 in AM / UC), the potency of which is preserved as disclosed in the specification and claims.

[0177] Dosage form

[0178] Provided below are dosage forms of the compositions described herein, including admixed compositions comprising fetal support tissue.

[0179] In some embodiments, the composition mixed as described herein is applied as an aqueous suspension. In some embodiments, the aqueous suspension includes water, Ringer's solution and / or isotonic sodium chloride solution. In some embodiments, Ringer's solution is lactated Ringer's saline. In some embodiments, the aqueous suspension includes sweeteners or flavorings, colorants or dyes and emulsifiers or suspending agents (if necessary), and also includes diluent water, ethanol, propylene glycol, glycerol or a combination thereof. In some embodiments, the aqueous suspension includes a suspending agent. In some embodiments, the aqueous suspension includes sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth gum and / or acacia gum. In some embodiments, the aqueous suspension includes a dispersant or a wetting agent. In some embodiments, aqueous suspensions include naturally occurring phospholipids, such as lecithin, or condensation products of alkylene oxides and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long-chain fatty alcohols, such as heptadecaethylene-oxyhexadecanol, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitol monooleate. In some embodiments, aqueous suspensions include preservatives. In some embodiments, aqueous suspensions include ethyl or n-propyl parabens. In some embodiments, aqueous suspensions include sweeteners. In some embodiments, aqueous suspensions include sucrose, saccharin or aspartame.

[0180] In some embodiments, the composition of the mixture described herein is applied in the form of an oily suspension. In some embodiments, the oily suspension is prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin). In some embodiments, the oily suspension comprises a thickener (e.g., beeswax, hard paraffin, or acetyl alcohol). In some embodiments, the oily suspension comprises a sweetener (e.g., the above-mentioned sweetener). In some embodiments, the oily suspension comprises an antioxidant (e.g., butylated hydroxyanisole or alpha-tocopherol).

[0181] In some embodiments, the mixed compositions described herein are formulated for administration by injection, such as parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered by intra-articular injection. In some embodiments, the mixed composition is administered as a sterile solution, suspension or emulsion. In some embodiments, the mixed composition is formulated for topical administration, local administration or inhalation. In some embodiments, the bioactive components (e.g., for example, HA, nHC-HA / PTX3 and other disclosed bioactive components) in the mixed compositions disclosed herein diffuse out from the mixed composition and enter the surrounding tissue. In some embodiments, the mixed fetal support tissue product formulated for topical administration also includes a penetration enhancer, a gelling agent, a patch, an adhesive, a softener or a combination thereof.

[0182] In some embodiments, formulations for injection are presented in unit dosage form, for example, in ampoules or multi-dose containers, with an added preservative.

[0183] In some embodiments, the mixed compositions described herein are formulated for topical topical administration. Topical topical preparations include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, films, rods, liposomes, microparticles, microspheres, lipid complexes, and nanoparticles. In some embodiments, topical topical preparations are applied by using a patch, a bandage, or a wound dressing. In some embodiments, mixed fetal tissue products, compositions, extracts, methods, or devices are formulated for treating or preventing tissue damage, such as ulcers, wounds, perforations, burns, surgeries, injuries, or fistulas. In some embodiments, the methods or compositions disclosed herein, etc., prevent or treat tissue necrosis or ischemic conditions. In some embodiments, ischemic conditions include cardiac ischemia, ischemic colitis, mesenteric ischemia, cerebral ischemia, acute limb ischemia, cyanosis, and gangrene, wherein the treatment method may include contacting the ischemic tissue with the mixed fetal support tissue product disclosed herein. In some embodiments, a method for treating a neurological condition in an individual in need is also provided herein, comprising contacting the ischemic tissue with a mixed fetal support tissue product.

[0184] In some embodiments, the mixed compositions described herein are formulated as compositions in the form of solids, cross-linked gels, or liposomes. In some embodiments, fetal tissue support products comprising nHC-HA / PTX3 complexes are formulated as insoluble cross-linked hydrogels. In some embodiments, the mixed fetal support tissue compositions are formulated as gels or topical surface preparations.

[0185] In some embodiments, the topical formulation includes a gelling agent (or thickening agent). Suitable gelling agents include, but are not limited to, cellulose, cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starches, tragacanth gum, carboxyvinyl polymers, carrageenan, paraffin, petrolatum, acacia gum (gum arabic), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, fucus, bentonite, carbomer, carrageenan, carbopol resin, xanthan gum, cellulose, microcrystalline cellulose (MCC), carob, carrageenan, dextrose, furcellaran, gelatin, gum ghatti, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, karaya gum, polyethylene glycol (e.g., PEG 200-4500), tragacanth, ethylcellulose, ethyl hydroxyethylcellulose, ethyl methylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxidized polygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinyl pyrrolidone (PVP: povidone), or a combination thereof.

[0186] In some embodiments, the topical surface formulation disclosed herein comprises an emollient. Emollients include, but are not limited to, castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, corn oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, chrysanthemum oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated monostearate glyceryl, hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, methyl palmitate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyl isostearate, methyl isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate Propyl lactate, lauryl lactate, myristyl lactate, and cetyl lactate, oleyl myristate, oleyl stearate, and oleyl oleate, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid, ricinoleic acid, arachidic acid, behenic acid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, sinapyl alcohol, 2-octyldodecanol, lanolin and lanolin derivatives, beeswax, spermaceti, myristyl myristate, stearyl stearate, carnauba wax, candelilla wax, lecithin, and cholesterol.

[0187] In some embodiments, the mixed compositions described herein are formulated with one or more natural polymers. In some embodiments, the mixed compositions described herein are formulated with natural polymers, and the natural polymers are fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate. In some embodiments, the mixed compositions described herein are formulated with polymer gels formulated with natural polymers. In some embodiments, the mixed compositions described herein are formulated with polymer gels formulated with natural polymers, and the natural polymers are, for example, but not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, and combinations thereof.

[0188] In some embodiments, the mixed fetal support tissue comprises a pharmaceutically acceptable excipient, carrier, or combination thereof. In some embodiments, the mixed fetal support tissue is formulated into a non-solid dosage form. In some embodiments, the mixed fetal support tissue is formulated into a solid dosage form.

[0189] In some embodiments, the compositions of mixing described herein are formulated for application to eyes or tissues associated therewith. Preparations suitable for application to eyes include but are not limited to solutions, suspensions (e.g., aqueous suspensions), ointments, gels, creams, liposomes, liposomes, pharmacoplasts, nanoparticles or combinations thereof. In some embodiments, the compositions of mixing described herein for topical application to eyes are applied by spraying, cleaning or a combination thereof. In some embodiments, the compositions of mixing described herein are applied to eyes by injectable long-acting formulations.

[0190] As used herein, a "depot formulation" is a controlled release formulation (e.g., subcutaneously, intramuscularly, intravitreally, or subconjunctivally) implanted into the eye or tissue associated therewith (e.g., sclera). In some embodiments, the depot formulation is prepared by forming a microencapsulated matrix (also referred to as a microencapsulated matrix) of the mixed composition described herein in a biodegradable polymer. In some embodiments, the depot formulation is prepared by encapsulating the mixed composition described herein in a liposome or microemulsion.

[0191] The preparation for application to the eye has an ophthalmologically acceptable osmotic pressure. In some cases, tears have an isotonic value comparable to a 0.9% sodium chloride solution. In some embodiments, an isotonic value comparable to about 0.6% to about 1.8% sodium chloride is suitable for topical surface application to the eye. In some embodiments, the preparation disclosed herein for application to the eye has an osmotic molar concentration of about 200 to about 600mOsm / L. In some embodiments, the preparation disclosed herein for application to the eye is hypotonic, and therefore any suitable substance needs to be added to reach an appropriate osmotic pressure range. The eye-acceptable substance for regulating osmotic pressure includes, but is not limited to, sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0192] The formulation for administration to the eye has an ophthalmologically acceptable clarity. Examples of ophthalmologically acceptable clarifying agents include, but are not limited to, polysorbate 20, polysorbate 80, or a combination thereof.

[0193] In some embodiments, the preparation for application to the eye includes an ophthalmologically acceptable viscosity enhancer. In some embodiments, the viscosity enhancer increases the time that the preparation disclosed herein stays in the eye. In some embodiments, the time that the preparation disclosed herein stays in the eye is increased to obtain greater drug absorption and effect. Non-limiting examples of mucoadhesive polymers include carboxymethyl cellulose, carbomer (acrylic acid polymer), poly (methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0194] In some embodiments, the composition is formulated for injection into the eye, for example, by injection into the tissue near the eye and the deeper tissue or connection space or tumor in the surrounding area or around the tumor. In some embodiments, the composition is administered by intravitreal injection into the eye. In some embodiments, the composition is administered by intraocular injection, subretinal injection, intravitreal injection, periocular administration, subconjunctival injection, retrobulbar injection, anterior chamber injection (including entering the anterior chamber or vitreous chamber) or sub-Tenon's injection. In some embodiments, the preparation for application to the eye is applied or delivered to the posterior segment of the eye (for example, to the retina, choroid, vitreous body and optic nerve). In some embodiments, the topical surface preparation disclosed herein for application to the eye is used for delivery to the posterior portion of the eye, and the topical surface preparation includes a solubilizing agent, for example, dextran sulfate and / or cyclodextrin. The glucan sulfates used in some embodiments include, but are not limited to, dextran sulfate, cyclodextrin sulfate, and β-1,3-glucan sulfate (both natural and its derivatives), or any compound that temporarily binds and can be retained on tissues containing fibroblast growth factor (FGF), which improves the stability and / or solubility of the drug, and / or improves the penetration and ocular absorption of the topical formulations disclosed herein for application to the eye. The cyclodextrin derivatives used as solubilizers in some embodiments include, but are not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, hydroxypropyl β-cyclodextrin, sulfated α-cyclodextrin, sulfated β-cyclodextrin, sulfobutyl ether β-cyclodextrin.

[0195] dose

[0196] The amount of the composition administered will depend in part on the individual being treated. Where the composition is administered to a human subject, the daily dosage will generally be determined by the prescribing physician, which will generally vary according to the individual's age, sex, diet, weight, general health and response, severity of individual symptoms, the exact disease or condition being treated, severity of the disease or condition being treated, time of administration, route of administration, handling of the composition, rate of excretion, drug combination, and the judgment of the prescribing physician.

[0197] In some embodiments, the fetal support tissue is in a freeze-dried and water-soluble form. Both the powder and liquid forms of the fetal support tissue contain nHC-HA / PTX3 complexes. To prepare the powder form, fresh or frozen fetal support tissue that has been screened for donor qualifications is cleaned, cut, soaked, and becomes a qualified starting material (RSM). Subsequently, under sterile room temperature conditions, the RSM undergoes freeze drying and micronization to grind into fine particles ( Figure 1AIn some embodiments, micronization can be performed with or without freeze drying to form a liquid or powder form. For the powder form, the micronized fetal support tissue is screened for particle size, then undergoes in-process control and quality control assessment to become a bulk drug substance that can be packaged and undergoes various terminal sterilization processes to become a finished drug product ( Figure 1B ). In some embodiments, the powdered tissue containing nHC-HA / PTX3 can be reconstituted in a suitable excipient, or the powder can be stored frozen at -80°C. In some embodiments, a liquid form of the fetal support tissue is prepared by rehydration after micronization. The liquid form of the fetal support tissue can also be prepared after cryo-pulverization. For example, in order to cryo-pulverize the fetal support tissue into a liquid form, the tissue is sterilized at 4°C ( Figure 2A-2C ). The fetal support tissue is cryo-pulverized, extracted, centrifuged, and filtered. In some embodiments, the cryo-pulverized tissue extract is centrifuged and filtered under different centrifugation and filtration parameters to control the particle size. In some embodiments, filtration is performed to sterilize the extract (filter pore size is equal to about to less than 0.2 um). After cryo-pulverizing the tissue, Figure 2A-2C The tissue is treated as shown; the tissue composition can be mixed, various dilutions can be made, and after the treatment control is completed, the extract containing nHC-HA / PTX3 is packaged as a finished drug. In some embodiments, the dosage of the HC-HA / PTX3 complex is between about 0.001 and about 1000 mg / kg body weight / day. In some embodiments, the amount of the HC-HA / PTX3 complex disclosed herein is in the range of about 0.5 to about 50 mg / kg / day. In some embodiments, the amount of the HC-HA / PTX3 complex disclosed herein is about 0.0001 to about 7 g / day. In some embodiments, the amount of the HC-HA / PTX3 complex disclosed herein is about 0.01 to about 7 g / day. In some embodiments, the amount of the HC-HA / PTX3 complex disclosed herein is about 0.02 to about 5 g / day. In some embodiments, the amount of the HC-HA / PTX3 complex disclosed herein is about 0.05 to about 2.5 g / day. In some embodiments, the amount of a HC-HA / PTX3 complex disclosed herein is from about 0.1 to about 1 g / day.

[0198] In some embodiments, a composition comprising an nHC-HA / PTX3 complex disclosed herein is administered before, during, or after an adverse change occurs in a tissue. In some embodiments, an nHC-HA / PTX3 composition disclosed herein is administered together with a combination therapy before, during, or after a disease or condition occurs. In some embodiments, the timing of administering the nHC-HA / PTX3 composition disclosed herein varies. Therefore, in some examples, the nHC-HA / PTX3 composition disclosed herein is used as a prophylactic and is continuously administered to a subject prone to adverse changes in a tissue to prevent adverse changes from occurring in the tissue. In some embodiments, the nHC-HA / PTX3 composition disclosed herein is administered to a subject as soon as possible during or after the adverse change occurs. In some embodiments, administration of the nHC-HA / PTX3 composition described herein begins within the first 48 hours after the occurrence of an adverse change, preferably within the first 48 hours after the onset of symptoms, more preferably within the first 6 hours after the onset of symptoms, and most preferably within 3 hours after the onset of symptoms. In some embodiments, the initial administration is by any feasible route, such as, for example, intravenous injection, bolus injection, 5 minutes to about 5 hours of infusion, pills, capsules, transdermal patches, oral buccal delivery, or a combination thereof. The nHC-HA / PTX3 compositions described herein are preferably administered as soon as possible after the adverse changes are detected or suspected to have occurred, and continue for the length of time required for treatment, for example, about 1 month to about 3 months. In some embodiments, the length of treatment varies from subject to subject, and the length is determined using known standards. In some embodiments, the compositions described herein are administered for at least 2 weeks, preferably about 1 month to about 5 years, and more preferably about 1 month to about 3 years.

[0199] In some embodiments, the nHC-HA / PTX3 compositions described herein are administered in a single dose, once daily. In some embodiments, the compositions described herein are administered in multiple doses, more than once daily. In some embodiments, the compositions described herein are administered twice daily. In some embodiments, the compositions described herein are administered three times daily. In some embodiments, the compositions described herein are administered four times daily. In some embodiments, the compositions described herein containing nHC-HA / PTX3 complexes are administered more than four times daily.

[0200] In the event that the individual's condition does not improve, at the physician's discretion, the nHC-HA / PTX3 compositions described herein are administered chronically (i.e., over an extended period of time, including throughout the individual's life) to ameliorate or otherwise control or limit the symptoms of the individual's disease or condition.

[0201] In some embodiments, when the individual's condition does improve, at the doctor's discretion, the nHC-HA / PTX3 complex disclosed herein is continuously administered, or the dose of the administered drug is temporarily reduced or temporarily stopped for a period of time (ie, a "drug holiday"). In some embodiments, the length of the drug holiday ranges from 2 days to 1 year, including (by way of example only) 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dose reduction during a drug holiday is 10%-100%, including by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0202] Once the individual's condition improves, a maintenance dose is administered as necessary. In some embodiments, the dose or dosing frequency, or both, is then reduced, depending on the symptoms, to a level that maintains the improved disease, condition, or disorder. In some embodiments, the individual requires long-term intermittent treatment upon any recurrence of symptoms.

[0203] In some embodiments, the pharmaceutical compositions described herein are unit dosage forms suitable for single administration of precise doses. In a unit dosage form, the preparation is divided into unit doses containing an appropriate number of nHC-HA / PTX3 complexes disclosed herein. In some embodiments, the unit dose is in a packaged form containing a discrete number of preparations. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-resealable containers. In some embodiments, a multi-dose resealable container is used, in which case a preservative is typically included in the composition. In some embodiments, preparations for parenteral injection are presented in unit dosage form, including but not limited to ampoules, or in multi-dose containers, and preservatives are added.

[0204] A daily dosage suitable for the nHC-HA / PTX3 complex disclosed herein is, for example, about 0.01 to 2.5 mg / kg body weight. The indicated daily dosage for large mammals (including but not limited to humans) is in the range of about 0.5 mg to about 100 mg, conveniently administered in divided doses, including but not limited to up to four times a day or in a sustained release form. Unit dosage forms suitable for oral administration include about 1 to 50 mg of active ingredient. The above ranges are only suggestive, as there are many variables associated with individual treatment regimens, and it is not uncommon for significant deviations from these recommended values. In some embodiments, the dosage varies according to many variables, which are not limited to the activity of the nHC-HA / PTX3 complex used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition to be treated, and the judgment of the practitioner.

[0205] In some embodiments, the compositions described herein are packaged as manufactured articles, which include packaging materials, effective prevention and / or treatment of a disease or disorder pharmaceutical composition, and a label indicating that the pharmaceutical composition is used to reprogram fibroblasts in tissues that have been adversely changed due to a disease or disorder. In some embodiments, the pharmaceutical composition is packaged into a unit dosage form, which includes the amount of the pharmaceutical composition for a single dose or multiple doses. In some embodiments, the packaged composition includes a freeze-dried powder of the pharmaceutical composition, which is reconstituted (e.g., with water or saline) before administration.

[0206] Medical devices and biomaterial compositions

[0207] In some embodiments, the compositions described herein are assembled directly on the surface of an implantable medical device or formulated as a coating for an implantable medical device. In some embodiments, the compositions described herein are assembled directly on the surface of an implantable medical device or a portion thereof.

[0208] Exemplary implantable medical devices include, but are not limited to, artificial joints, orthopedic devices, bone implants, contact lenses, sutures, surgical staples, surgical clips, catheters, angioplasty balloons, sensors, surgical instruments, electrodes, needles, syringes, wound drains, shunts, urethral inserts, metal or plastic implants, heart valves, artificial organs, abdominal bands, annuloplasty rings, guide wires, K-wires or Denham pins, stents, stent grafts, vascular grafts, pacemakers, pellets, wafers, medical tubing, infusion cannulas, implantable defibrillators, neurostimulators, glucose sensors, cerebrospinal fluid shunts, implantable drug pumps, spinal cages, artificial intervertebral discs, ocular implants, cochlear implants, breast implants, nucleus pulposus replacement devices, ear tubes, intraocular lenses, drug delivery systems, microparticles, nanoparticles, and microcapsules.

[0209] In some embodiments, the method of using a composition, product or extract or a composition, product or extract of a fetal support tissue comprising various forms of mixing as described herein relieves pain, reduces body inflammation and reduces scarring. In certain embodiments, such methods of use or such compositions, products or extracts promote regenerative healing. In some embodiments, regenerative healing includes, for example, nerve regeneration or articular cartilage regeneration and various body regenerative healing. In some embodiments, nerve regeneration inhibits sensitization / stimulation. In some embodiments, disclosed herein may be a method of using a composition, or a composition, product or extract comprising, for example, a granular material from cryopreserved amniotic membrane (AM) and umbilical cord (UC), wherein the mixed fetal support product, composition or extract can be used for various clinical applications, including, for example, knee osteoarthritis, facet joint osteoarthritis, plantar fasciitis, chronic wound healing, complex wound healing, and foot pad injury healing.

[0210] In some embodiments, complex wounds may be wounds with chronic body ulcers, ischemic wounds, or include exposed bones, muscles, tendons, joint capsules, bone loss (e.g., due to necrosis of soft tissue, bones, etc.) and may be difficult to heal, increasing the sensitivity of skin, muscle, tendon infection. In some embodiments, patients with complex wounds may be susceptible to bone infection, including osteomyelitis. In some embodiments, the mixed fetal support tissue extracts of the present disclosure may be used to treat complex wounds to achieve healing by revascularization, re-epithelialization, and rekeratinization from the peripheral edge of the wound to the granulation tissue. In some embodiments, complex wounds may be correction of congenital defects, for example, intrauterine spina bifida repair using the present disclosure. In other embodiments, the present disclosure may be used to treat complex wounds caused by diabetes, diabetic neuropathy, such as complex gangrenous wounds that may require amputation, and wherein the use of the present disclosure after sharp debridement, bone resection, bone biopsy, and / or open cortex, etc., may completely cancel amputation processing (e.g., amputation of gangrenous feet, toes, limbs, etc.).

[0211] In some embodiments, a method of using a composition or product or a composition or product including, for example, a cryopreserved amniotic membrane (AM) and umbilical cord (UC) of mixed fetal support tissue (including, for example, clarix flo and cryopreserved AM compositions) can lead to faster recovery of mechanical hyperalgesia and reduce thermal hyperalgesia. In certain embodiments, the combination of micronized and freeze-dried AM and UC can be a mixed fetal support tissue product, composition or extract, which contains any sheet and non-sheet form of fetal support tissue products disclosed herein, and wherein such a composition or a method of using a composition can be used as a drug or treatment for any clinical indication disclosed herein. In some embodiments, any administration method disclosed herein can be used to administer such a drug, treatment method or its method of use to relieve physical pain, injury, inflammation, scarring, infection or disease. In some embodiments, the composition described herein is directly assembled on a scaffold, microparticle, microcapsule or microcarrier for delivering a biomaterial (e.g., stem cell or insulin-producing cell). In some embodiments, the composition described herein is attached to a microcapsule or directly assembled on a microcapsule.

[0212] How to use

[0213] In certain embodiments, disclosed herein is a method of using the compositions described herein by the methods described herein. In some embodiments, the composition comprises a therapeutically effective amount of a mixed composition comprising fetal support tissue. In some embodiments, the composition comprises a therapeutically effective amount of a mixed composition comprising micronized fetal support tissue. In some embodiments, the composition comprises a therapeutically effective amount of a mixed composition comprising fragmented fetal support tissue. In some embodiments, the composition comprises a therapeutically effective amount of a mixed composition comprising micronized fetal support tissue. In some embodiments, the fetal support tissue is placental amnion (PAM) or substantially separated PAM, umbilical cord amnion (UCAM) or substantially separated UCAM, chorion or substantially separated chorion, amnion-chorion or substantially separated amnion-chorion, placenta or substantially separated placenta, umbilical cord or substantially separated umbilical cord, or any combination thereof. In some embodiments, the fetal support tissue produced by the methods disclosed herein includes fetal support tissue and a pharmaceutically acceptable carrier. In some embodiments, the fetal support tissue product disclosed herein is formulated for administration by topical topical administration or injection. In some embodiments, the fetal support tissue products disclosed herein are formulated as a solution, suspension, or emulsion.

[0214] In some embodiments, the mixed compositions disclosed herein are used to inhibit at least one of the following: scarring, inflammation, adhesions, and angiogenesis, but may promote angiogenesis. In some embodiments, a mixed fetal support tissue product, such as, for example, a product containing an isolated nHC-HA / PTX3 composition, is used to inhibit the regrowth of cancer cells in a tumor, including contacting the area surrounding the tumor with an isolated nHC-HA / PTX3 complex after a surgical procedure, thereby inhibiting the regrowth of cancer cells in the area surrounding the tumor. In some cases, a method of killing cancer cells in a tumor includes contacting a tumor or a surrounding area with an isolated nHC-HA / PTX3 complex before, during, or after a surgical procedure, thereby killing cancer cells. In some embodiments, a mixed composition, product, or extract containing isolated nHC-HA / PTX3 can be used to inhibit, treat, or as a method of treating or reducing epithelial cell proliferation, cell migration, or epithelial-mesenchymal transition (EMT) in an individual in need thereof, wherein such a composition or treatment method comprises administering to the individual a therapeutically effective amount of the mixed composition or product and a pharmaceutically acceptable diluent, excipient, vehicle, or carrier, thereby preventing or reducing epithelial cell proliferation, cell migration, or EMT. In some embodiments, the mixed compositions, extracts, or products disclosed herein, regardless of form, can be administered as a method of treating, medicating, or inhibiting cancer cell regeneration in a tumor of an individual in need thereof, wherein such treatment, medicating, or treating method comprises contacting the area surrounding the tumor with an isolated nHC-HA / PTX3 complex after a surgical procedure, thereby inhibiting the regrowth of cancer cells in the area surrounding the tumor.

[0215] A method of killing cancer cells in a tumor of an individual in need thereof, comprising contacting the tumor or a peritumoral area with an isolated nHC-HA / PTX3 complex before, during, or after a surgical procedure, thereby killing the cancer cells.

[0216] In some embodiments, fetal tissue products, compositions such as, for example, nHC-HA / PTX3, can be used as bone implants comprising an outer surface coated with substantially isolated nHC-HA / PTX3 complexes.

[0217] In some embodiments, the mixed compositions disclosed herein can be used to promote wound healing. In some embodiments, mixed fetal support tissue products (such as, for example, nHC-HA / PTX3 compositions) are used to promote angiogenesis of tissues comprising endothelial cells and pericytes, including reprogramming pericytes to a first progenitor cell phenotype by contacting tissue with fetal support tissue products, and reprogramming endothelial cells to a second progenitor cell phenotype by contacting tissue with fetal support tissue products. In some embodiments, this healing that promotes angiogenesis relates to treating ischemic tissues comprising endothelial cells and pericytes, including reprogramming pericytes to a first progenitor cell phenotype by contacting tissue with fetal support tissue products, and reprogramming endothelial cells to a second progenitor cell phenotype by contacting tissue with fetal support tissue products. In some embodiments, the use is a mixed use. In some embodiments, the mixed fetal compositions disclosed herein can be used to promote neural regeneration. In some embodiments, the methods disclosed herein include using mixed compositions, extracts, or products to promote, repair, rebuild, replace, enhance, or supplement soft tissues that are damaged or atrophied in individuals in need. In some embodiments, damaged or atrophic soft tissue is contacted with the product, extract, or composition to reduce or prevent epidural fibrosis, adhesions, or scarring after spinal surgery in an individual in need. In some embodiments, fetal support tissue products can be used to treat a condition or disease, including: contacting fibroblasts in a tissue affected by the condition in a subject in need with a composition comprising soluble nHC-HA / PTX3 for a period of time sufficient to reprogram the fibroblasts into cells with different phenotypes, thereby treating the condition or disease. In some embodiments, the methods and / or compositions comprising the mixed fetal support tissue products disclosed herein inhibit osteoclast differentiation, bone resorption, bone formation, bone remodeling, treatment of alveolar bone degeneration, and treatment of Paget's disease. In some embodiments, the methods or compositions disclosed herein, devices prepared from mixed fetal support tissues (including preparations of mixed tissue extracts), and products containing nHC-HA / PTX3 are formulated and administered for the treatment of bone tumors, or for the treatment of disease conditions or disorders defined by a lack or defect in bone formation, or for the treatment of osteoporosis, or for administration of treatment as an implant or patch, wherein the patch or implant is placed on an osteolytic bone or osteolytic joint of an individual in need. In some embodiments, the composition is formulated into an orthopedic prosthesis. In some embodiments, mixed fetal support tissue products, extracts, compositions, or methods may be administered to promote bone healing, or to inhibit diseases or conditions characterized by bone damage, bone defects, or bone deficiency. In some embodiments, the mixed compositions disclosed herein undergo minimal manipulation. In some embodiments, the mixed compositions disclosed herein do not include other products except water, crystalloids, or sterilizing agents, preservatives, or storage agents.In some embodiments, the mixed compositions disclosed herein do not have systemic effects, and their primary function is not dependent on the metabolic activity of living cells.

[0218] In some embodiments, the mixed composition or product includes mixed fetal support tissue, such as umbilical cord amniotic membrane (UCAM) obtained from a fresh, frozen or previously frozen umbilical cord, and a preservation medium, wherein substantially all cells of the UCAM are dead, wherein the UCAM is not dehydrated. In some embodiments, the mixed composition or product from mixed fetal support tissue includes a method of repairing, reconstructing, replacing, enhancing or supplementing damaged or atrophic soft tissue in an individual in need thereof, comprising contacting the damaged or atrophic soft tissue of the individual with an umbilical cord product from which water has not been removed.

[0219] In some embodiments, the mixed fetal support tissue product or composition can be used as a method of reducing or preventing epidural fibrosis, adhesions, or scarring following spinal surgery in an individual in need thereof, wherein the method comprises contacting the spinal surgical site with the umbilical cord product.

[0220] In some embodiments, the mixed fetal support tissue product can be used as a composition, product or method for treating ocular wounds or repairing damaged ocular tissue in an individual in need. In some embodiments, the mixed compositions, products or extracts disclosed herein can be used as a drug, treatment or method for treating or preventing individuals with proliferative vitreoretinopathy (PVR) or preventing PVR in individuals who have experienced retinal detachment, including administering to individuals with PVR or who have experienced retinal detachment, wherein the treatment may include administering a substantially isolated native heavy chain hyaluronic acid / pentameric protein 3 complex (HC-HA / PTX3), reconstructed HC-HA / PTX3 or a combination thereof using the methods disclosed herein.

[0221] In some embodiments, the mixed compositions, products, extracts disclosed herein may be a medicament, treatment or method for treating or preventing proliferative vitreoretinopathy (PVR) in an individual suffering from PVR or preventing PVR in an individual who has experienced retinal detachment, wherein the mixed product is administered to an individual suffering from PVR or who has experienced retinal detachment as disclosed herein, wherein a substantially isolated native HC-HA / PTX3 product and an additional therapeutic agent for treating or preventing PVR are provided. In the present disclosure, providing a therapeutically effective amount is understood to mean any dose or treatment capable of alleviating a condition, providing relief or a cure, according to any and existing procedures or doses believed to provide therapeutic or preventive effectiveness for the treatment, prevention or relief of distress, pain or condition provided.

[0222] In some embodiments, the mixed compositions disclosed herein are used as coverings (e.g., wound coverings). In some embodiments, the use is a mixed use. In some embodiments, the mixed compositions undergo minimal manipulation. In some embodiments, the mixed compositions do not include other products other than water, crystalloids or sterilizing agents, preservatives, or storage agents. In some embodiments, the mixed compositions do not have systemic effects and their primary function does not rely on the metabolic activity of living cells.

[0223] In some embodiments, the mixed compositions disclosed herein are used to promote wound repair. In some embodiments, the use is a mixed use. In some embodiments, the mixed compositions undergo minimal manipulation. In some embodiments, the mixed compositions do not include other products other than water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed compositions do not have systemic effects and their primary function does not rely on the metabolic activity of living cells.

[0224] In some embodiments, the mixed compositions disclosed herein are used as adhesion barriers. In some embodiments, the mixed compositions undergo minimal manipulation. In some embodiments, the mixed compositions do not contain other products other than water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed compositions do not have systemic effects, and their primary function does not depend on the metabolic activity of living cells.

[0225] Repair and replenish damaged tissue

[0226] In some embodiments, the mixed compositions disclosed herein are used as wound coverings or for promoting wound repair. In some embodiments, the mixed compositions undergo minimal manipulation. In some embodiments, the mixed compositions do not include other preparations except water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed compositions do not have systemic effects, and their primary function does not rely on the metabolic activity of living cells.

[0227] In some embodiments, the tissue is damaged, restricted or lost due to injury (e.g., burns; surgical incisions; necrotic areas caused by infection, trauma or toxins; lacerations). In some embodiments, the tissue is damaged, restricted or lost due to burns. In some embodiments, the tissue is damaged, restricted or lost due to wounds (e.g., incisions, lacerations, abrasions). In some embodiments, the tissue is damaged, restricted or lost due to necrosis. In some embodiments, the tissue is damaged, restricted or lost due to ulcers. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously or a combination thereof.

[0228] In some embodiments, the mixed compositions disclosed herein contain HA, nHC-HA / PTX3 complexes, and various bioactive factors that promote tissue repair, which are present in the matrix of, for example, AM / UC and contain anti-angiogenesis and anti-inflammatory proteins. In some embodiments, the HA, nHC-HA / PTX3 complexes in the mixed compositions disclosed herein diffuse out of the mixed compositions and enter the surrounding tissues.

[0229] burn

[0230] In some embodiments, the mixed compositions disclosed herein are applied to burns. In some embodiments, the mixed compositions disclosed herein are applied to first degree burns. In some embodiments, the mixed compositions disclosed herein are applied to second degree burns. In some embodiments, the mixed compositions disclosed herein are applied to third degree burns. In some embodiments, the mixed compositions are applied to a substrate prior to being placed on the burn.

[0231] Wound

[0232] In some embodiments, the mixed composition disclosed herein is applied to a wound (e.g., incision, laceration, abrasion, ulcer, puncture, penetration) of the skin. In some embodiments, the mixed composition is applied to a substrate before being placed on the wound. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravesical, subconjunctival, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0233] In some embodiments, the mixed compositions disclosed herein are applied to incisions in organs (e.g., skin, brain, stomach, kidney, liver, intestines, lungs, bladder, trachea, esophagus, vagina, ureters, and blood vessel walls). In some embodiments, the mixed compositions disclosed herein are applied to surgical incisions. In some embodiments, the mixed compositions disclosed herein are applied to colectomy sites. In some embodiments, the mixed compositions disclosed herein are applied to gastrectomy sites. In some embodiments, the mixed compositions disclosed herein are applied to breast surgery sites (e.g., breast reduction surgery, breast augmentation surgery, and mastectomy). In some embodiments, the mixed compositions disclosed herein are applied to substrates before being placed on a wound.

[0234] In some embodiments, the mixed compositions disclosed herein are used as a covering for skin incisions (e.g., incisions of the epidermis, dermis, and / or subcutaneous tissue). In some embodiments, the mixed compositions disclosed herein are used for skin repair or supplementation after hemorrhoid surgery. In some embodiments, the mixed compositions are applied to a substrate before being placed on a wound.

[0235] Necrosis

[0236] In some embodiments, the composition of mixing disclosed herein is used as a protective graft in a necrotic tissue area (e.g., from infection). In some embodiments, the composition of mixing disclosed herein is used as a protective graft on a necrotic skin area. In some embodiments, the composition of mixing disclosed herein is placed on a necrotic tissue area. In some embodiments, the composition of mixing is applied to a substrate before being placed on necrotic tissue. In some embodiments, the composition of mixing is applied by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the composition of mixing is epidural, intrathecal, by suction, intravenous or a combination thereof.

[0237] ulcer

[0238] In some embodiments, the mixed compositions disclosed herein are used as protective coverings on ulcers. In some embodiments, the mixed compositions are applied to substrates before being placed on ulcers. In some embodiments, the mixed compositions are administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed compositions are administered epidurally, intrathecally, by inhalation, intravenously or in combination thereof.

[0239] In some embodiments, the ulcer is a foot ulcer (e.g., a diabetic foot ulcer or an arterial insufficiency ulcer). In some embodiments, treating a foot ulcer comprises (a) preparing a wound (e.g., cleaning a wound); and (b) placing a mixed composition disclosed herein on the wound. In some embodiments, treating a foot ulcer comprises (a) preparing a wound (e.g., cleaning a wound); (b) placing a mixed composition disclosed herein on the wound; and (c) covering the mixed composition with a protective barrier (e.g., a silver cell dressing, a methib, a gauze, or a bandage). In some embodiments, the mixed composition is applied to a substrate before being placed on the ulcer.

[0240] In some embodiments, the ulcer is a venous stasis (VS) ulcer. In some embodiments, treating a VS ulcer comprises (a) preparing the wound (e.g., debridement of the wound); and (b) placing a mixed composition disclosed herein on the wound. In some embodiments, treating a VS ulcer comprises (a) preparing the wound (e.g., debridement of the wound); (b) placing a mixed composition disclosed herein on the wound; and (c) covering the mixed composition with a protective barrier (e.g., a wound cover, an antimicrobial dressing, gauze, or a bandage). In some embodiments, the mixed composition is applied to a substrate prior to being placed on the wound.

[0241] In some embodiments, the ulcer is a corneal ulcer (i.e., ulcerative keratitis). In some embodiments, treating a corneal ulcer comprises (a) preparing a wound (e.g., cleaning a wound); and (b) placing a mixed composition disclosed herein on the wound. In some embodiments, treating a corneal ulcer comprises (a) preparing a wound (e.g., cleaning a wound); (b) placing a mixed composition disclosed herein on the wound; and (c) covering the mixed composition with a protective barrier (e.g., a contact lens or a bandage). In some embodiments, the mixed composition is applied to a substrate before being placed on the wound.

[0242] Soft tissue use

[0243] In certain embodiments, disclosed herein is the use of a mixed composition for repairing, reconstructing, replacing or supplementing a recipient's damaged, limited or lost soft tissue (e.g., tendon). In certain embodiments, a mixed composition, product or extract in sheet form or non-sheet form disclosed herein can be used to treat or as a method of reprogramming cells having a first phenotype, comprising contacting a cell having a first phenotype with a composition comprising soluble nHC-HA / PTX3 for a period of time sufficient to reprogram a cell having a first phenotype to a second cell having a second phenotype. In some embodiments, the mixed composition can be used to treat or can be used as a method for treating a disorder or disease of a subject in need thereof, comprising contacting a fibroblast in a tissue affected by the disorder of a subject in need thereof with a composition comprising soluble nHC-HA / PTX3 for a period of time sufficient to reprogram the fibroblast to a cell having a different phenotype, thereby treating the disorder or disease. In certain embodiments, any form of the mixed compositions, extracts, or products disclosed herein may be used as a method of treatment, medicine, or regenerating tissue, comprising contacting cells having a first differentiated phenotype with a composition comprising soluble nHC-HA / PTX3 for a period of time sufficient to reprogram the cells having the first differentiated phenotype into progenitor cells having a progenitor cell phenotype, and differentiating the progenitor cells into cells having a second differentiated phenotype, thereby regenerating tissue.

[0244] In some embodiments, the use is a mixed use. In some embodiments, the mixed composition undergoes minimal manipulation. In some embodiments, the mixed composition does not include other preparations except water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed composition does not have systemic effects and its primary function does not rely on the metabolic activity of living cells.

[0245] In some embodiments, the mixed compositions disclosed herein include HA, nHC-HA / PTX3 complexes, and various bioactive factors that promote tissue repair, which are present in the matrix of, for example, AM / UC and contain anti-angiogenesis and anti-inflammatory proteins. In some embodiments, the HA, nHC-HA / PTX3 complexes in the mixed compositions disclosed herein diffuse out of the mixed compositions and enter the surrounding tissues.

[0246] In some embodiments, the mixed compositions disclosed herein are used as a covering on a soft tissue incision (e.g., eyelids form a tissue plane between different layers of soft tissue). In some embodiments, the mixed compositions are applied to a substrate and then used as a covering on a soft tissue incision (e.g., eyelids form a tissue plane between different layers of soft tissue). In some embodiments, the mixed compositions are administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed compositions are administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0247] In some embodiments, the mixed fetal support tissue compositions / extracts, methods, kits, devices, etc. disclosed herein are useful treatments or therapeutic agents for bladder conditions. In some embodiments, the bladder condition can be a bladder disorder, syndrome, disease, and / or infection. For example, the bladder condition can be, for example, urinary incontinence. For example, the bladder condition can be, for example, bladder cancer. In some embodiments, the bladder condition can be related to cancer or tumor therapy, such as therapy for lower intestinal or urinary tract cancer. In some embodiments, the present disclosure can be administered in the bladder soft tissue of patients suffering from interstitial cystitis, overactive bladder, or other lower urinary tract neurourinary function conditions.

[0248] In some embodiments, the mixed compositions disclosed herein are used as structural (tectonic) supports for soft tissues.

[0249] In some embodiments, the mixed compositions disclosed herein prevent adhesions in joint or tendon repair.

[0250] In some embodiments, the mixed compositions disclosed herein are used to repair tendons or joints (e.g., rotator cuff repair, Achilles tendinopathy, and hand tendon repair). In some embodiments, the mixed compositions disclosed herein are used to reinforce tendons or joints. In some embodiments, the mixed compositions disclosed herein are used to prevent adhesion of the healing tendon to surrounding tissues, tendons, or joints. In some embodiments, the mixed compositions disclosed herein are used to prevent scar tissue from forming on a tendon.

[0251] In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used to strengthen the smaller tendons and ligaments of the foot and ankle, including the posterior tibial tendon, the peroneal tendon, the flexor tendon and the extensor tendon and the ligament of the lateral complex of the ankle. In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used to strengthen the primary repair of the quadriceps tendon and the patellar tendon around the knee joint. In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a periosteal patch of the bone transplant in joint replacement. In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used to strengthen the hip joint and knee capsule tissue of the defect after the total joint repair surgery.

[0252] In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to repair the torn rotator cuff. In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as a patch on rotator cuff muscle or tendon (for example, suprascapularis tendon). In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to rebuild rotator cuff muscle or tendon (for example, suprascapularis tendon). In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to strengthen rotator cuff muscle or tendon (for example, suprascapularis tendon). In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to strengthen rotator cuff muscle or tendon (for example, suprascapularis tendon). In some embodiments, the composition of mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to prevent soft tissue from adhering to rotator cuff muscle or tendon (for example, suprascapularis tendon).

[0253] In some embodiments, the mixed composition disclosed herein is used to repair gums. In some embodiments, the mixed composition disclosed herein is used to repair gum atrophy. In some embodiments, the mixed composition disclosed herein is applied to a substrate and used as a patch on the gums. In some embodiments, the mixed composition disclosed herein is applied to a substrate and used as a patch on an exposed root surface. In some embodiments, the mixed composition disclosed herein is used to rebuild gums. In some embodiments, the mixed composition disclosed herein is used to strengthen gums. In some embodiments, the mixed composition disclosed herein is used to strengthen gums. In some embodiments, the mixed composition disclosed herein is used to prevent soft tissue from adhering to gums.

[0254] In some embodiments, the mixed composition described herein is applied to substrate, and the substrate / mixed composition is used as a protective graft at the fascia incision or tear. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used as the structure (construction) support of fascia. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used as a substitute or supplement of fascia. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used to repair hernia (for example, repair fascia). In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used to repair inguinal hernia. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used to repair femoral hernia. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used to repair umbilical hernia. In some embodiments, the mixed composition disclosed herein is applied to substrate, and the substrate / mixed composition is used to repair incisional hernia. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used to repair a diaphragmatic hernia. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used to repair a Cooper's hernia, an epigastric hernia, a hiatal hernia, a diverticulum hernia, a lumbar hernia, a retrograde intestinal loop incarceration hernia, an obturator hernia, a pantaloon hernia, a paraesophageal hernia, a paraumbilical hernia, a perineal hernia, an extraperitoneal hernia, an incomplete intestinal wall hernia, a sliding hernia, a sciatic hernia, a spigelian hernia, a sports hernia, a Velpeau hernia, or an inguinal hernia containing an appendix (Amyand's hernia).

[0255] In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used to repair intervertebral disc herniation. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as a protective graft at the intervertebral disc incision or tear. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as a protective graft at the fiber ring incision or tear. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as the structure (construction) support of the intervertebral disc. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as the structure (construction) support of the fiber ring. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as a substitute or supplement of the intervertebral disc. In some embodiments, the composition of the mixing disclosed herein is applied to substrate, and the composition of substrate / mixing is used as the structure (construction) support of the intervertebral disc. In some embodiments, a mixed composition disclosed herein is applied to a substrate and the substrate / mixed composition is used as a replacement or supplement for the annulus fibrosus.

[0256] In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used on brain incisions, or in one (or all) of the meninges (i.e., dura mater, pia mater, and / or arachnoid). In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as supports for one (or all) of the meninges (i.e., dura mater, pia mater, and / or arachnoid) structures (constructions). In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as substitutes for one (or all) of the meninges (i.e., dura mater, pia mater, and / or arachnoid).

[0257] In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used on an incision of the lung or pleura. In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a structural (construction) support of the pleura. In some embodiments, the composition of the mixing disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a substitute for the pleura.

[0258] In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used on the incision of the tympanic membrane. In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as structural (structural) supports for tympanic membranes. In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as substitutes for tympanic membranes.

[0259] In some embodiments, the mixed compositions disclosed herein are applied to a substrate, and the substrate / mixed compositions are used as a protective graft on a heart or pericardial incision. In some embodiments, the mixed compositions disclosed herein are applied to a substrate, and the substrate / mixed compositions are used as a structural (constructural) support for the pericardium. In some embodiments, the mixed compositions disclosed herein are applied to a substrate, and the substrate / mixed compositions are used as a substitute for the pericardium.

[0260] In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as protective grafts on peritoneal incisions. In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as structural (construction) supports of peritoneum. In some embodiments, the mixed compositions disclosed herein are applied to substrates, and the substrate / mixed compositions are used as substitutes for peritoneum.

[0261] Eye Uses

[0262] In certain embodiments, disclosed herein are compositions disclosed herein for repairing, reconstructing, replacing or supplementing damaged, limited or lost ocular tissue of a recipient. In some embodiments, the mixed compositions are administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed compositions are administered epidurally, intrathecally, by inhalation, intravenously or a combination thereof.

[0263] In some embodiments, the use is a mixed use. In some embodiments, the mixed composition undergoes minimal manipulation. In some embodiments, the mixed composition does not include other preparations except water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed composition disclosed herein does not have systemic effects, and its primary function does not rely on the metabolic activity of living cells.

[0264] In some embodiments, the mixed compositions disclosed herein include HA, nHC-HA / PTX3 complexes, and various bioactive factors that promote tissue repair, which are present in the matrix of, for example, AM / UC and contain anti-angiogenesis and anti-inflammatory proteins. In some embodiments, the HA, nHC-HA / PTX3 complexes in the mixed compositions disclosed herein diffuse out of the mixed compositions and enter the surrounding tissues.

[0265] Treatment of glaucoma

[0266] As used herein, "glaucoma" refers to a condition characterized by loss of retinal ganglion cells in the optic nerve. In some cases, glaucoma is caused in part or in whole by elevated intraocular pressure in the anterior chamber (AC). Changes in intraocular pressure depend on the production of the liquid aqueous humor by the ciliary processes of the eye and its drainage through the trabecular meshwork.

[0267] Glaucoma drainage devices (GDDs) are medical devices implanted in the eye that relieve intraocular pressure by providing an alternative pathway for aqueous humor to drain. If left uncovered, GDD tubes can corrode and leave the eye susceptible to intraocular infection. Therefore, there is a need to cover GDD tubes. Currently, patches used to cover GDD tubes are made from pericardium, sclera, and cornea. These patches are approximately 400-550 microns thick. The thinness of these patches causes them to melt by 25% within 2 years, potentially exposing the shunt tube again.

[0268] In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the GDD tube is covered with the substrate / mixed composition. In some embodiments, the thickness of the substrate / mixed composition is 300-600 microns. In some embodiments, the substrate / mixed composition will not melt 25% within 2 years. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously or a combination thereof.

[0269] Treatment of eye ulcers

[0270] In some embodiments, a mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used to cover epithelial defects and / or ulcers in the eye. In some embodiments, the mixed composition is applied to patients with defects or ulcers caused by dry eye, graft-versus-host disease, Graves' disease, traumatic corneal injury, corneal surgery, or neurotrophic keratopathy. The present disclosure can be applied in conjunction with contact lenses, such as, but not limited to, bandage contact lenses (BCL) or scleral lenses. The effectiveness of combination therapy has been demonstrated, for example, by administering the Clarix / Neox Flo product in conjunction with BCL, which can be used to treat subjects with mild to moderate dry eye or for situations where the subject is difficult to accept standard care. In this case, the Flo-BCL combination can be applied for 2 to 3 days. In some embodiments, the present disclosure can be reconstituted in saline and applied to the concave surface of a BCL, further applied or applied to the corneal surface of a subject with mild to moderate dry eye (e.g., graded as DEWS1-2 (DEWS-International Dry Eye Symposium)), the subject may exhibit superficial corneal epithelial keratopathy and / or remain symptomatic after receiving standard care. In some embodiments, the present disclosure can be administered in conjunction with a BCL, wherein the BCL can be a brand that has been approved by an internationally recognized agency (e.g., the U.S. Federal Drug Administration (FDA)). For example, the BCL can be any brand, such as ACUVUE AIR NIGHT and AQUA, Bausch and Lomb Bausch and Lomb 2 and other brands. In certain embodiments, the present disclosure is reconstructed in saline and applied to the concave surface of the BCL, and can be retained on the corneal surface of the object for a period of not less than 3, 4, or 5 days. In certain embodiments, the length of the period can be 5 days or longer. In some embodiments, the treatment is placed on the cornea of ​​the object for at least 1, 2, 3, 4, or 5 days. In some embodiments, the fetal support tissue product may include a fragmented product, which may be a product or composition containing mixed or non-mixed fetal support tissue. For example, the fragmented product may include, for example, a mixed fetal support tissue containing amniotic membrane and umbilical cord tissue, and the mixed fetal support tissue has become a fragmented, centrifuged, sterile-filtered fetal support tissue product / extract or composition (hereinafter referred to as a fragmented product or TTBT01 drug). In some embodiments, the TTBT01 drug may include fetal support tissue from multiple donors and / or donor batches. In some embodiments, the TTBT01 drug may include fetal support tissue from multiple donors and / or donor batches. In some embodiments, the TTB01 drug or fragmented product can be packaged in a single-use unit-dose vial. In some embodiments, the TTBT01 drug or fragmented product can be topically applied to the ocular surface of the PROSE or BostonSight scleral lens concave surface of a subject with DEWS grade 3-4 severe dry eye. PROSE and BostonSight scleral lenses are Class II FDA510(k) approved medical devices. This product combining TTBT01 with a selected scleral lens can be applied to the subject as specified in the user guide. For example, TTBT01 can be filled in a scleral lens daily and applied to the eye for at least 4 hours. In some embodiments, the TTBT01 drug can be filled into a scleral lens every 1 day, 2 days or more days and applied to the eye for the length of time specified by a trained ophthalmologist or trained medical staff (doctor). For optimal results, the TTBT01 drug can be continuously applied to the scleral lens, and during continuous treatment, for example, it can be applied daily for 5-7 days. In other cases, administration may be less than 5 days or more than 7 days. In general, the drug in the scleral lens can be reconstituted daily and administered as is, or determined according to the doctor's instructions and the response of the subject being treated. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof. In some embodiments, the substrate is a bandage contact lens or a scleral lens.

[0271] In some embodiments, the bottom of the ulcer is debrided with a surgical sponge, and the epithelium with poor adhesion near the edge of the ulcer is removed (for example, the block of the eye where the adjacent epithelium becomes very adherent). In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is transferred to the recipient's eye. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and then the substrate / mixed composition is fixed to the eye by a suture (for example, an interrupted 10-0 nylon suture or a continuous 10-0 nylon suture), and the suture knot is buried. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is fixed to the eye using fibrin glue. In some embodiments, a protective layer is applied to the mixed composition / substrate or the entire eye (for example, a contact lens). In some embodiments, the substrate / mixed composition also includes antibiotics (for example, neomycin, polymyxin b sulfate and dexamethasone).

[0272] In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used for conjunctival, sclera, eyelid and orbital rim surface reconstruction. In some embodiments, the damage to the conjunctival surface is caused by symblepharon lysis; surgical removal of tumors, lesions and / or scar tissue; excimer laser photorefractive keratectomy and therapeutic keratectomy; or a combination thereof.

[0273] Coronary Use

[0274] In certain embodiments, disclosed herein is the use of the mixed nHC-HA / PTX3 (complex) compositions disclosed herein to repair, reconstruct, replace, or supplement damaged, restricted, or lost coronary tissue of a recipient. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0275] In some embodiments, the use is a mixed use. In some embodiments, the mixed composition undergoes minimal manipulation. In some embodiments, the AM does not include other preparations other than water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed composition does not have systemic effects and its primary function does not depend on the metabolic activity of living cells.

[0276] In some embodiments, the mixed compositions disclosed herein include HA, nHC-HA / PTX3 complexes, and various bioactive factors that promote tissue repair, which are present in the matrix of, for example, AM / UC and contain anti-angiogenesis and anti-inflammatory proteins. In some embodiments, the HA, nHC-HA / PTX3 complexes in the mixed compositions disclosed herein diffuse out of the mixed compositions and enter the surrounding tissues.

[0277] Coronary artery bypass grafting

[0278] Disclosed herein is the use of the mixed nHC-HA / PTX3 (complex) compositions described herein in coronary artery bypass grafting. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is transplanted to a coronary artery to bypass an arterial block characterized by atherosclerosis. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0279] Heart valves

[0280] In some embodiments, the mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a substrate, and the substrate / mixed composition is applied to a heart valve. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a structural (structural) support for a heart valve. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a substitute for a heart valve. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0281] Veins and arteries

[0282] In some embodiments, the mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a vein or artery. In some embodiments, the mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a substrate, and the substrate / mixed composition is applied to a vein or artery. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a structural (construction) support for a vein or artery. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0283] Neurological Uses

[0284] In some embodiments, the mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a nerve or neural tissue. In some embodiments, the mixed nHC-HA / PTX3 (complex) composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a covering on a nerve (e.g., a peripheral nerve). In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a covering on a nerve transplant, a nerve transplant, or a repair nerve. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a covering on a nerve (e.g., a peripheral nerve) incision. In some embodiments, the mixed composition disclosed herein is applied to a substrate, and the substrate / mixed composition is used as a structural (structural) support for a nerve (e.g., a peripheral nerve). In some embodiments, the mixed composition disclosed herein prevents adhesion in nerve repair.

[0285] In certain embodiments, disclosed herein is the use of a mixed nHC-HA / PTX3 (complex) composition for repairing, reconstructing, replacing or supplementing damaged, restricted or lost neural tissue of a recipient. Various diseases are associated with neural pain or neural damage. Without limitation in any way, exemplary conditions associated with neural pain include pain in the musculoskeletal system or pain in the locomotor system of a mammal, which may include bones, muscles and connective tissue, and may include tendons, cartilage, and ligaments. Musculoskeletal pain may be caused by short-term injuries or lifelong conditions, or by conditions that may lead to persistent functional limitations, disability, and pain, such as strains, repeated overuse, sprains, dislocations, fractures, or bone or joint deformities, which may include scoliosis, spinal curvature, or a combination thereof. In some embodiments, musculoskeletal conditions may affect joints, including conditions such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, ankylosing spondylitis, and the like. In some embodiments, musculoskeletal conditions may affect bones, including conditions such as osteoporosis, osteopenia, and related fragility fractures, traumatic fractures, and amputations. In some embodiments, musculoskeletal diseases may affect muscles, such as sarcopenia, or pain in the spine (e.g., back and neck). In some embodiments, musculoskeletal pain or conditions may affect multiple body regions or body systems and may result in, for example, localized body or systemic pain, widespread pain conditions, inflammatory diseases (including connective tissue diseases) and vasculitis (including systemic lupus erythematosus), bursitis, and other conditions that may be caused by pain, infection, or injury to the musculoskeletal system. In some embodiments, the mixed compositions are administered to treat conditions including neuropathic pain (e.g., musculoskeletal pain).

[0286] In some embodiments, the mixed nHC-HA / PTX3 (complex) composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0287] In some embodiments, the use is a mixed use. In some embodiments, the mixed composition undergoes minimal manipulation. In some embodiments, the mixed composition does not contain other preparations except water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed composition does not have systemic effects and its primary function does not rely on the metabolic activity of living cells.

[0288] Disclosed herein are processes for generating fetal support tissue while retaining the biological activity of the fetal support tissue, including components of the fetal support tissue, such as HA, HC-HA / PTX3 growth factors, cytokines, and other active biological components, wherein the potency of the HC-HA / PTX3 can be monitored to ensure its adequate preservation, as disclosed in the specification and claims. In some embodiments, nHC-HC / PTX3 and other biologically active components found in the mixed compositions disclosed herein diffuse from the mixed compositions into surrounding tissues.

[0289] In some embodiments, the mixed nHC-HA / PTX3 (complex) compositions disclosed herein are applied to a substrate and the substrate / mixed composition is used as a non-contractile wrap for injured nerves. In some embodiments, the mixed compositions described herein prevent or minimize scarring, encapsulation, chronic compression, nerve entrapment, and nerve entrapment. In some embodiments, the mixed compositions described herein prevent or minimize neuroma formation. In some embodiments, the mixed compositions described herein prevent or minimize the migration of endogenous growth factors (e.g., nerve growth factor) present during nerve repair.

[0290] Spine Uses

[0291] Disclosed herein, in certain embodiments, are the use of the mixed compositions described herein during spinal surgery.

[0292] In some embodiments, the mixed composition described herein is used during laminectomy. In some embodiments, the use is a mixed use. In some embodiments, the mixed composition undergoes minimal manipulation. In some embodiments, the mixed composition does not include other preparations except water, crystalloids or sterilizing agents, preservatives or storage agents. In some embodiments, the mixed composition does not have systemic effects and its primary function does not rely on the metabolic activity of living cells.

[0293] In some embodiments, the mixed compositions disclosed herein include HA, nHC-HA / PTX3 complexes, and various bioactive factors that promote tissue repair, which are present in, for example, the matrix of AM / UC and contain anti-angiogenesis and anti-inflammatory proteins. In some embodiments, the HA, nHC-HA / PTX3 complexes in the mixed compositions disclosed herein diffuse out of the mixed compositions and enter the surrounding tissues. In some embodiments, the mixed compositions are administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0294] In some embodiments, the mixed compositions described herein are used to reduce or prevent epidural fibrosis and / or scar adhesions after spinal surgery (e.g., laminectomy). In some embodiments, after spinal surgery (e.g., laminectomy), the mixed compositions described herein are implanted between the dura mater and the overlying tissue. In some embodiments, after spinal surgery (e.g., laminectomy), implanting the mixed compositions described herein between the dura mater and the overlying tissue can reduce or prevent the migration of fibroblasts to the dura mater and the deposition of collagen on the dura mater.

[0295] In some embodiments, the mixed compositions described herein are used to reduce or prevent hypertrophic scar formation after spinal surgery (e.g., laminectomy). In some embodiments, the mixed compositions described herein are used to reduce or prevent the formation of epidural / peridural / perineural scars after surgery (e.g., laminectomy). In some embodiments, the mixed compositions described herein are used to reduce or prevent hypertrophic scar formation after spinal surgery (e.g., laminectomy). In some embodiments, the mixed compositions disclosed herein are used to reduce or prevent the development of membranes after laminectomy.

[0296] In some embodiments, the mixed compositions described herein are used to reduce or prevent epidural compression or dural tethering after spinal surgery (e.g., laminectomy). In some embodiments, the mixed compositions described herein are used to reduce or prevent nerve root tethering after spinal surgery (e.g., laminectomy). In some embodiments, the mixed compositions described herein are used to reduce or prevent arachnoiditis after spinal surgery (e.g., laminectomy).

[0297] In some embodiments, the mixed composition disclosed herein further comprises crushed bone tissue. In some embodiments, the mixed composition comprising fragmented bone tissue disclosed herein is used during spinal fusion surgery. In some embodiments, the mixed composition comprising fragmented bone tissue disclosed herein is implanted between adjacent vertebrae. In some embodiments, implanting the mixed composition comprising fragmented bone tissue disclosed herein between two adjacent vertebrae can promote vertebral fusion.

[0298] In some embodiments, the composition of mixing disclosed herein is used as a protective graft on dura mater incision. In some embodiments, the composition of mixing disclosed herein is applied to substrate, and substrate / mixed composition is used as a structure (construction) support of dura mater. In some embodiments, the composition of mixing disclosed herein is applied to substrate, and substrate / mixed composition is used as a substitute of dura mater.

[0299] Other uses of the mixed composition

[0300] In some embodiments, the mixed nHC-HA / PTX3 (complex) compositions disclosed herein are applied to a patch or wound dressing. In some embodiments, the mixed composition is administered by parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the mixed composition is administered epidurally, intrathecally, by inhalation, intravenously, or a combination thereof.

[0301] In some embodiments, the mixed compositions disclosed herein are used as dermal fillers. In some embodiments, the mixed compositions disclosed herein are injected into facial tissues beneath the dermis. In some embodiments, the mixed compositions disclosed herein are injected under facial wrinkles and aging lines (e.g., nasolabial folds, lip lines, "crow's feet" and forehead wrinkles). In some embodiments, the mixed compositions disclosed herein are used for lip augmentation. In some embodiments, the mixed compositions disclosed herein are injected into the lips. In some embodiments, the mixed compositions disclosed herein are used after or in combination with skin regeneration treatments (e.g., laser ablation treatments).

[0302] In some embodiments, the mixed compositions disclosed herein are used to treat arthritis (e.g., osteoarthritis, rheumatoid arthritis, septic arthritis, ankylosing spondylitis, spondylosis). In some embodiments, the mixed compositions disclosed herein are injected into arthritic joints (e.g., knee joints).

[0303] In some embodiments, the mixed nHC-HA / PTX3 (complex) compositions disclosed herein are used to inhibit bone resorption in individuals in need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or bone tumors. In some embodiments, the mixed composition is injected into the joint. In some embodiments, the mixed composition is in contact with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the mixed composition is coated with a bone scaffold, a bone implant, or a bone prosthesis (e.g., an osseointegration implant). As used herein, "osseointegration implant" refers to a three-dimensional implant comprising a hole into which osteoblasts and supporting connective tissue can migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee joint or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

[0304] In some embodiments, the mixed composition disclosed herein is used to promote or induce bone formation in an individual in need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease (Paget'sdisease) or bone tumor. In some embodiments, the mixed composition is injected into the joint. In some embodiments, the mixed composition is in contact with the bone (for example, by using a wound dressing or a bandage). In some embodiments, the mixed composition is coated with a bone scaffold, a bone implant or a bone prosthesis (for example, an osseointegration implant). As used herein, "osseointegration implant" refers to a three-dimensional implant comprising a hole in which osteoblasts and supporting connective tissue can migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee joint or joint. In some embodiments, the bone scaffold is placed in a fracture site. In some embodiments, the bone scaffold is expandable or retractable.

[0305] In some embodiments, the mixed composition disclosed herein is used to inhibit osteoclast differentiation. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease (Paget's disease) or bone tumor. In some embodiments, the mixed composition is injected into the joint. In some embodiments, the mixed composition is in contact with the bone (for example, by using a wound dressing or a bandage). In some embodiments, the mixed composition is coated with a bone scaffold, a bone implant or a bone prosthesis (for example, an osseointegration implant). As used herein, "osseointegration implant" refers to a three-dimensional implant comprising a hole, into which osteoblasts and supporting connective tissue can migrate. In some embodiments, a bone scaffold is inserted into the intramedullary canal of a bone. In some embodiments, a bone scaffold is placed in the tarsal sinus. In some embodiments, a bone scaffold is placed in a knee joint or a joint. In some embodiments, a bone scaffold is placed at a fracture. In some embodiments, a bone scaffold is expandable or retractable.

[0306] In some embodiments, the mixed composition disclosed herein is used to promote osteoblast mineralization in individuals in need. In some embodiments, individuals suffer from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease (Paget'sdisease) or bone tumors. In some embodiments, the mixed composition is injected into the joint. In some embodiments, the mixed composition is in contact with the bone (for example, by using a wound dressing or a bandage). In some embodiments, the mixed composition is coated with a bone scaffold, a bone implant or a bone prosthesis (for example, an osseointegration implant). As used herein, "osseointegration implant" refers to a three-dimensional implant comprising a hole in which osteoblasts and supporting connective tissue can migrate. In some embodiments, a bone scaffold is inserted into the intramedullary canal of a bone. In some embodiments, a bone scaffold is placed in the tarsal sinus. In some embodiments, a bone scaffold is placed in a knee joint or a joint. In some embodiments, a bone scaffold is placed at a fracture. In some embodiments, a bone scaffold is expandable or retractable.

[0307] In some embodiments, the mixed compositions disclosed herein are used to balance the bone resorption and bone formation of individuals in need. In some embodiments, individuals suffer from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease (Paget'sdisease) or bone tumors. In some embodiments, the mixed compositions are injected into the joint. In some embodiments, the mixed compositions are in contact with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the mixed compositions are coated with bone scaffolds, bone implants or bone prostheses (e.g., osseointegration implants). As used herein, "osseointegration implants" refer to three-dimensional implants comprising osteoblasts and supporting connective tissues to which holes can migrate. In some embodiments, the bone scaff...

Claims

1. A composition comprising mixed fetal support tissue, wherein the mixed fetal support tissue comprises a therapeutically effective amount of a native HC-HA / PTX3 complex.

2. The composition of claim 1, wherein the mixed fetal support tissue improves the homogeneity of the native HC-HA / PTX3 complex in the composition.

3. The composition of claim 1 or 2, wherein the mixed fetal support tissue comprises fetal support tissue from multiple donors.

4. The composition of claim 1 or 2, wherein the mixed fetal support tissue comprises fetal support tissue from at least three donors.

5. The composition of claim 1 or 2, wherein the mixed fetal support tissue comprises fetal support tissue from at least five donors.

6. The composition of claim 1 or 2, wherein the mixed fetal support tissue comprises fetal support tissue from at least ten donors.

7. The composition of any one of claims 1 to 6, wherein the composition exhibits therapeutic efficacy as determined by an assay comprising one or more of a HA content assay, a PTX3 content assay, a BCA total protein assay, an ODI-TRAP assay, an M2 polarization assay, or a WST-1 assay.

8. The composition of claim 7, wherein the therapeutic efficacy is determined by the ODI-TRAP assay.

9. The composition of claim 8, wherein the composition exhibits statistically significant inhibition of TRAP activity compared to a positive control in the ODI-TRAP assay when the HA content of the composition is at least 200 μg / ml, 250 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1,000 μg / ml.

10. The composition of claim 9, wherein the significant inhibition of TRAP activity is at least 70% inhibition of TRAP activity.

11. The composition of claim 9, wherein the significant inhibition of TRAP activity is at least 80% inhibition of TRAP activity.

12. The composition of claim 9, wherein the significant inhibition of TRAP activity is at least 85% inhibition of TRAP activity.

13. The composition according to any one of claims 9 to 12, wherein the HA content of the composition is at least 200 μg / ml.

14. The composition according to any one of claims 9 to 12, wherein the HA content of the composition is at least 250 μg / ml.

15. The composition according to any one of claims 9 to 12, wherein the HA content of the composition is at least 300 μg / ml.

16. The composition according to any one of claims 9 to 12, wherein the HA content of the composition is at least 350 μg / ml.

17. The composition of any one of claims 9 to 12, wherein the HA content of the composition is about 300 μg / ml.

18. The composition of claim 9, wherein the positive control is RANKL treatment.

19. The composition of claim 7, wherein the therapeutic efficacy is determined by the M2 assay.

20. The composition of claim 7, wherein the therapeutic efficacy is determined by a NO assay.

21. The composition of claim 7, wherein the therapeutic efficacy is determined by the WST-1 assay.

22. The composition of claim 7, wherein the therapeutic efficacy is determined by BCA protein assay.

23. The composition of claim 7, wherein the therapeutic efficacy is determined by HA content assay.

24. The composition according to any one of claims 1 to 23, wherein the HA content of the composition is at least 75 μg / ml.

25. The composition of any one of claims 1 to 24, wherein the HA content of the composition is at least 90 μg / ml.

26. The composition of any one of claims 1 to 25, wherein the total protein content of the composition is at least 250 μg / ml.

27. The composition of any one of claims 1 to 26, wherein the mixed fetal support tissue is freeze-dried.

28. The composition of any one of claims 1 to 27, wherein the mixed fetal support tissue is freeze-dried prior to mixing the fetal support tissue.

29. The composition of any one of claims 1 to 28, wherein the mixed fetal support tissue is freeze-dried after mixing the fetal support tissue.

30. The composition of any one of claims 1 to 29, wherein the mixed fetal support tissue composition has been frozen or cryopreserved.

31. The composition of any one of claims 1 to 30, wherein the mixed fetal support tissue comprises a particle size of about 0.01 micrometers (μm) to about 240 μm in diameter.

32. The composition of any one of claims 1 to 30, wherein the mixed fetal support tissue comprises an average particle size of about 0.5 μm.

33. The composition of any one of claims 1 to 32, wherein the mixed fetal support tissue comprises placenta, umbilical cord, placental amnion, umbilical cord amnion, Wharton's jelly, chorion, or amnion-chorion, or any combination thereof.

34. The composition of any one of claims 1 to 32, wherein the mixed fetal support tissue comprises placenta amnion, umbilical cord, or both.

35. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is comminuted prior to mixing.

36. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is comminuted after mixing.

37. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is fragmented.

38. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is filtered or ultrafiltered.

39. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is decellularized.

40. The composition of any one of claims 1 to 34, wherein the mixed fetal support tissue is devitalized.

41. The composition of any one of claims 1 to 40, wherein the mixed fetal support tissue is a gel extract.

42. The composition of any one of claims 1 to 40, wherein the mixed fetal support tissue is in powdered form.

43. The composition of any one of claims 1 to 42, wherein the mixed fetal support tissue comprises 5% or more water by weight.

44. The composition of any one of claims 1 to 43, wherein the mixed fetal support tissue comprises up to 15% water by weight.

45. The composition of any one of claims 1 to 44, wherein the mixed fetal support tissue is obtained from frozen or once frozen fetal support tissue.

46. ​​The composition of any one of claims 1 to 45, wherein the mixed fetal support tissue is substantially free of veins or arteries.

47. A composition according to any one of claims 1 to 46, wherein substantially all cells of the mixed fetal support tissue are dead.

48. The composition of any one of claims 1 to 47, wherein the mixed fetal support tissue is terminally sterilized, gamma irradiated, filtered, e-beam sterilized, or a combination thereof.

49. A composition according to any one of claims 1 to 48, wherein the composition is provided as a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

50. The composition of claim 49, wherein the pharmaceutically acceptable excipient is normal saline.

51. The composition of any one of claims 1 to 50, wherein the mixed fetal support tissue composition exhibits reduced variation in native HC-HA / PTX3 complex activity compared to a non-mixed fetal support tissue composition as determined by an ODI-TRAP assay, a BCA assay, or a HA quantification assay.

52. The composition of claim 51, wherein the reduced variation is a coefficient of variation of 10% or less.

53. The composition of claim 51, wherein the reduced variation is a coefficient of variation of 5% or less.

54. The composition of claim 51, wherein the reduced variation is a coefficient of variation of 4% or less.

55. The composition of claim 51, wherein the reduced variation is a coefficient of variation of about 3%.

56. The composition of claim 51, wherein the reduced variation is a coefficient of variation of about 2% or less.

57. A method of treating a degenerative disease in a subject, the method comprising administering to the subject the mixed fetal support tissue of any one of claims 1 to 56.

58. A method of treating inflammation in a subject, the method comprising administering to the subject the mixed fetal support tissue of any one of claims 1 to 56.

59. A method of promoting wound healing in a subject, the method comprising administering to the subject the mixed fetal support tissue of any one of claims 1 to 56.

60. A method of treating osteoarthritis in a subject, the method comprising administering to the subject the mixed fetal support tissue of any one of claims 1 to 56.

61. The method of claim 60, wherein the osteoarthritis is osteoarthritis of the knee.

62. A method of preparing a composition according to any one of claims 1 to 56, comprising obtaining fetal support tissue from a plurality of donors and combining the fetal support tissue from the plurality of donors.

63. The method of claim 62, wherein the fetal support tissue from the plurality of donors is freeze-dried prior to said mixing.

64. The method of claim 62, wherein the fetal support tissue from the plurality of donors is freeze-dried after the mixing.

65. The method of any one of claims 62 to 64, further comprising processing the fetal support tissue from the plurality of donors by fragmentation, pulverization, micronization, or a combination thereof.

66. The method of any one of claims 62 to 65, further comprising devitalizing the fetal support tissue from the plurality of donors.

67. The method of any one of claims 62 to 65, further comprising decellularizing the fetal support tissue from the plurality of donors.

68. The method of any one of claims 62 to 67, wherein the fetal support tissue from the plurality of donors is not dehydrated.

69. The method of any one of claims 62 to 68, wherein the fetal support tissue from the plurality of donors is not dehydrated to contain less than 20% water by weight.

70. The method of any one of claims 62 to 68, wherein the fetal support tissue from the plurality of donors is not dehydrated to contain less than 15% water by weight.

71. A method for validating a mixed fetal support tissue product, the method comprising performing a validation assay on the mixed fetal support tissue, wherein the validation assay determines the amount and / or therapeutic efficacy of a native HC-HA / PTX3 complex.

72. The method of claim 71, wherein the native HC-HA / PTX3 has therapeutic activity.

73. The method of claim 71 or 72, wherein the validation assay comprises one or more of a HA content assay, a PTX3 content assay, an ODI-TRAP assay, a BCA protein assay, an M2 polarization assay, a NO assay, or a WST-1 assay.

74. The method of any one of claims 71 to 73, wherein the validation assay comprises an ODI-TRAP assay.

75. The method of claim 74, wherein the mixed fetal support tissue product exhibits statistically significant inhibition of TRAP activity compared to a positive control in the ODI-TRAP assay when the HA content of the composition is at least 200 μg / ml, 250 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1,000 μg / ml.

76. The method of claim 75, wherein the positive control is RANKL treatment.

77. The method of claim 71, wherein the therapeutic efficacy is determined by the M2 assay.

78. The method of claim 71, wherein the therapeutic efficacy is determined by a NO assay.

79. The method of claim 71, wherein the therapeutic efficacy is determined by the WST-1 assay.

80. The method of claim 71, wherein the therapeutic efficacy is determined by a BCA protein assay.

81. The method of claim 71, wherein the therapeutic efficacy is determined by HA content measurement.

82. The method of claim 71, wherein the therapeutic efficacy is determined by a PTX3 content assay.

83. The method of claim 75, wherein the HA content of the composition is at least 75 μg / ml.

84. The method of claim 75, wherein the HA content of the composition is at least 90 μg / ml.

85. The method of any one of claims 62 to 75, wherein the total protein content of the composition is at least 250 μg / ml.

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