Compositions and methods of making placental tissue laminates

Abstract

Disclosed herein are compositions and methods directed to preserved placental tissue samples. Methods provided herein can include forming a placental tissue laminate and dehydrating the placental tissue laminate. Compositions provided herein can include dehydrated placental tissue laminates. Also disclosed herein are methods of using dehydrated placental tissue laminates for treating wounds or damaged tissue, comprising dehydrated placental tissue laminates to the wound or damaged tissue.

Description

DESCRIPTIONCOMPOSITIONS AND METHODS OF MAKING PLACENTAL TISSUE LAMINATESCROSS-REFERENCE WITH RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 611,563, filed December 18, 2023, the contents of which is incorporated into the present application by reference.FIELD

[0002] The present disclosure generally relates to placental products, methods of processing placental tissues and / or making placental products, and use of placental products in the field of wound management and / or tissue repair. In some aspects, the disclosure concerns compositions comprising amniotic tissue laminates with exposed epithelial layers and differing surface roughness and / or porosities that exhibit reduced delamination, and methods of making and / or using the same.BACKGROUND

[0003] A wound is a disruption of the structure and function of tissue. A dermal wound involves the disruption of the skin and associated soft tissue architecture. Dermal wounds can be partial or full thickness wounds. They can also be acute wounds, chronic wounds, or bums (which can be acute or chronic). Wounds may also include, without limitation: internal organ wounds; mucous membrane wounds; vascular tissue wounds; soft tissue wounds including wounds to ligaments, tendons, and cartilage; and bone wounds.

[0004] The use of placental tissues for bums and other types of wounds is known. Placental tissues contain components that can be helpful for wound healing or tissue regeneration. These components can include extracellular matrix (ECM), growth factors, and cells, including mesenchymal stem cells (MSCs) that are responsible for orchestrating the healing process in different tissue types.

[0005] Issues with using placental tissues to treat wounds, however, include maintaining the functional and / or physical properties of placental tissues during and after storage and handling the tissues after storage and during use. To help with healing, the wound bed can be free of devitalized tissue, be well vascularized, and be moist. Wound dressings can help facilitate the wound healing process if they provide an environment to support the healingprocess by allowing cellular migration, vascular ingrowth, and / or the formation of granulation tissues. Many of the commercially available wound dressings do not have these capabilities to support the healing process, and some are not very effective at this. Additionally, some of these wound dressings are not cost-efficient or manageable for the medical practitioner.

[0006] Most human placental tissue products are thin and comprise a single layer. When surgeons or other medical practitioners apply the human placental tissue product to a wound or tissue for wound or tissue management or repair, the placental tissue is thin and can be difficult to control after rehydration, leading to sub-optimal handling properties. Available human placental tissue products that do comprise more than one layer do not specify how the tissue is to be layered and result in products having side-specific placement requirements. For example, surgeons or other medical practitioners may be required to monitor which side of the placental tissue product is being applied to the wound or tissue for optimal wound or tissue management or repair, which can increase the time required for wound or tissue management procedures.

[0007] One purpose of tissue preservation is to retain the viability and structural and functional characteristics of components of fresh tissue, such as the extracellular matrix (ECM), growth factor repertoire, and viable endogenous cells, while providing an extended shelf-life for tissues compared to fresh tissues. Preservation methods for placental tissue products, including placental tissue laminate products, include refrigeration, dehydration (e.g., lyophilization or air-drying), and cryopreservation. However, each of these methods suffer from drawbacks. Refrigeration of fresh tissues can maintain cell viability for only a short time, which can lead to a short shelf-life (weeks) and limited availability. Cryopreservation can retain living tissue cells for an extended time (months to years), but the cost and effort required to maintain ultra- low temperatures (i.e., -40° C or below) across the entire supply chain can limit utilization. Dehydration of tissues can provide an extended shelf-life (years) and can retain tissue matrix but can lead to tissue devitalization, which can negatively impact tissue biological function. Certain dehydration methods can improve shelf-life while not requiring ultra-low temperatures in the supply chain, but these advantages can come with unwanted effects associated with the dehydration process, such as but not limited to, increased tissue cracking and / or delamination of the dual placental tissue layers. This may increase waste and manufacturing costs, as cracked or delaminated placental tissue products may be discarded and replaced.SUMMARY

[0008] A discovery has been made that provides a solution to at least one or more of the aforementioned problems associated with manufacturing and using placental tissues (e.g., chorion, amnion, a chorion and amniotic membrane (e.g., amnio-chorion), Wharton’s jelly, umbilical cord, decidua, placental cotyledons or combinations thereof). In one aspect, provided herein are methods of manufacturing a placental tissue graft, the methods comprising (a) obtaining one or more placental tissues (e.g., amniotic tissues), each placental tissue having a first layer (e.g., a stromal layer) opposite a second layer (e.g., an epithelial layer), and forming a placental tissue laminate by (1) folding one of the one or more placental tissues to contact the first layer of a first portion of the placental tissue with the first layer of a second portion of the placental tissue; or (2) layering a first of the placental tissues with a second of the placental tissues by contacting the first layer of the first of the placental tissues with the first layer of the second of the placental tissues. In this way, a placental tissue graft (e.g., an amniotic tissue graft) is produced having a layer of a first type (e.g., an epithelial layer) external to each side the graft and layers of a second type (e.g., a stromal layer) internal to the graft. In some aspects, this configuration advantageously utilizes the sticky and gelatinous characteristic of the stromal layer. In some aspects, orientating the placental tissue layers in this manner can increase the binding affinity between the tissue layers compared to grafts having alternative layering arrangements (e.g., an epithelial layer facing a stromal layer or a stromal layer external to each side of the graft and epithelial layers internal to the graft). This can reduce or prevent delamination of the placental tissue layers, in some cases, even after the rehydration.

[0009] The placental tissue grafts may be manufactured by lyophilizing or air-drying the placental tissue laminate. In some aspects, lyophilizing or air-drying the placental tissue laminate can provide grafts having different characteristics, including different surface roughness and / or different tissue porosity, that render the grants useful for different wound healing applications. For example, in some aspects, lyophilized placental tissue laminates can have a surface roughness and / or tissue porosity to provide grafts that are ideal for wound protection, while air-dried placental tissue laminates can have a surface roughness and / or tissue porosity to provide grafts that are ideal for soft tissue repair. In some aspects, increasing thickness by of the placental tissue grafts by layering the placental tissues does not affect the roughness and / or tissue porosity of the grafts. In some aspects, an outer-facing epithelial layer provides a superior wound covering or wound barrier compared to an outer-facing stromal layer.

[0010] Accordingly, in certain aspects, as exemplary non-limiting benefits, methods of manufacturing a placental tissue graft described herein can provide at least one of, anycombination of, or all of the following features: (1) versatile placental tissue laminates that can be reversibly applied at wound sites, thereby avoiding possibly time-consuming and error- prone side-specific placement requirements; (2) thicker placental tissue laminates with more strongly adhered layers to improve handling properties and that retain structures similar to fresh tissue and living cells (e.g., preserved placental tissues that do not comprise substantive levels of cracks and / or delamination, or provide reduced relative levels of cracks and / or delamination relative to preserved placental tissues produced according to traditional preservation protocols); (3) placental tissue laminates that can be manufactured to optimize tissue porosity and / or surface roughness, depending on the method by which the placental tissue laminate grafts are prepared, for different wound healing applications; (4) placental tissue laminates having an extended shelf-life (e.g., months to years) without ultra-low supply chain temperatures to provide extended shelf-life; and / or (5) placental tissue laminates that do not comprise unwanted characteristics (e.g., cracking or delamination) after preservation, such that fewer products are discarded and replaced. Such compositions and methods would have numerous applications, such as clinical and / or commercial uses similar to uses of fresh intact tissues, while providing an extended shelf-life for tissues compared to fresh tissue and an environment conducive for regenerating healthy tissue and improving clinical outcomes.

[0011] Disclosed herein, in some aspects, a method of manufacturing an amniotic tissue graft, the method comprising: obtaining one or more amniotic tissues, each amniotic tissue comprising a stromal layer and an epithelial layer opposite the stromal layer; and forming an amniotic tissue laminate by: folding one of the one or more amniotic tissues to contact the stromal layer of a first portion of the amniotic tissue with the stromal layer of a second portion of the amniotic tissue; or layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the stromal layer of the first of the amniotic tissues with the stromal layer of the second of the amniotic tissues. In some aspects, the method further comprises inserting or layering a reinforcing material between the stromal layers of the amniotic tissue(s). In some aspects, the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or bio-synthetic material. In some aspects, the method further comprises sterilizing the amniotic tissue laminate.

[0012] In some aspects, the method further comprises lyophilizing the amniotic tissue laminate. In some aspects, the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for wound treatment. In some aspects, the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a burn, a thermal bum, a chemical bum, a partial thickness burn, ora full thickness burn; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum.

[0013] In some aspects, the method further comprises air-drying the amniotic tissue laminate. In some aspects, the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for soft tissue in need of repair. In some aspects, the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint.

[0014] Disclosed herein, in some aspects, is an amniotic tissue graft comprising a lyophilized amniotic tissue laminate, the lyophilized amniotic tissue laminate comprising: an amniotic tissue that has been folded to contact a stromal layer of a first portion of the amniotic tissue with a stromal layer of a second portion of the amniotic tissue; or a first amniotic tissue layered with a second amniotic tissue by contacting a stromal layer of the first amniotic tissue with a stromal layer of the second amniotic tissue. In some aspects, the amniotic tissue graft further comprises a reinforcing material inserted or layered between the stromal layers of the amniotic tissue(s). In some aspects, the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or bio-synthetic material. In some aspects, the amniotic tissue laminate is sterilized. In some aspects, the amniotic tissue laminate is lyophilized. In some aspects, the amniotic tissue laminate is airdried.

[0015] Disclosed herein, in some aspects, is a method of treating a wound, the method comprising topically applying to the wound an amniotic tissue graft prepared according to a method as disclosed herein or a lyophilized amniotic tissue graft as disclosed herein. In some aspects, the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a burn, a thermal bum, a chemical burn, a partial thickness bum, or a full thickness bum; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum.

[0016] Disclosed herein, in some aspects, is a method of repairing soft tissue, the method comprising applying to soft tissue in need of repair an amniotic tissue graft prepared according to a method as disclosed herein or a lyophilized amniotic tissue graft as disclosed herein. Insome aspects, the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint.

[0017] Also disclosed herein are the following Aspects 1-20. Aspect 1 is a method of manufacturing an amniotic tissue graft, the method comprising: obtaining one or more amniotic tissues, each amniotic tissue comprising a stromal layer and an epithelial layer opposite the stromal layer; and forming an amniotic tissue laminate by: folding one of the one or more amniotic tissues to contact the stromal layer of a first portion of the amniotic tissue with the stromal layer of a second portion of the amniotic tissue; or layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the stromal layer of the first of the amniotic tissues with the stromal layer of the second of the amniotic tissues. Aspect 2 is the method of Aspect 1, further comprising inserting or layering a reinforcing material between the stromal layers of the amniotic tissue(s). Aspect 3 is the method of Aspect 2, wherein the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or bio-synthetic material. Aspect 4 is the method of any one of Aspects 1-3, further comprising lyophilizing the amniotic tissue laminate. Aspect 5 is the method of Aspect 4, wherein the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for wound treatment. Aspect 6 is the method of Aspect 5, wherein the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a bum, a thermal bum, a chemical bum, a partial thickness burn, or a full thickness bum; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum. Aspect 7 is the method of Aspect 1, further comprising air-drying the amniotic tissue laminate. Aspect 8 is the method of Aspect 7, wherein the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for soft tissue in need of repair. Aspect 9 is the method of Aspect 8, wherein the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint. Aspect 10 is the method of any one of Aspects 1-9, further comprising sterilizing the amniotic tissue laminate.

[0018] Aspect 11 is an amniotic tissue graft comprising a lyophilized amniotic tissue laminate, the lyophilized amniotic tissue laminate comprising: an amniotic tissue that has been folded to contact a stromal layer of a first portion of the amniotic tissue with a stromal layer ofa second portion of the amniotic tissue; or a first amniotic tissue layered with a second amniotic tissue by contacting a stromal layer of the first amniotic tissue with a stromal layer of the second amniotic tissue. Aspect 12 is the tissue graft of Aspect 11, further comprising a reinforcing material inserted or layered between the stromal layers of the amniotic tissue(s). Aspect 13 is the tissue graft of Aspect 12, wherein the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or bio-synthetic material. Aspect 14 is the tissue graft of any one of Aspects 11-13, wherein the amniotic tissue laminate is sterilized. Aspect 15 is the tissue graft of any one of Aspects 11-14, wherein the amniotic tissue laminate is lyophilized. Aspect 16 is the tissue graft of any one of Aspects 11- 14, wherein the amniotic tissue laminate is air-dried.

[0019] Aspect 17 is a method of treating a wound, the method comprising topically applying to the wound an amniotic tissue graft prepared according to the method of any one of Aspects 1-10 or the lyophilized amniotic tissue graft of Aspect 15. Aspect 18 is the method of Aspect 17, wherein the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a burn, a thermal burn, a chemical bum, a partial thickness burn, or a full thickness bum; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum.

[0020] Aspect 19 is a method of repairing soft tissue, the method comprising applying to soft tissue in need of repair an amniotic tissue graft prepared according to the method of any one of Aspects 1-10 or the air-dried amniotic tissue graft of Aspect 16. Aspect 20 is the method of Aspect 19, wherein the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cmciate ligament (ACL), posterior cmciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint.

[0021] The disclosed materials, compositions, and components may be used for, may be used in conjunction with, may be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these materials may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, for example, if a class of components A, B, and C are disclosed as well as a class of components D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individuallyand collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0022] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0023] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0025] FIG. 1 shows an exemplary photo illustrating exemplary lyophilized and air-dried placental tissue dressings in accordance with aspects of the present disclosure.

[0026] FIG. 2 shows exemplary photos illustrating an exemplary amniotic tissue laminate graft in which the stromal layer of each amniotic tissue component is internal to the graft and the epithelial layer of each amniotic tissue component is external to the graft.

[0027] FIG. 3 shows exemplary photos illustrating an exemplary lyophilized amniotic tissue laminate graft in which the stromal layer of each amniotic tissue component is internal to the graft and the epithelial layer of each amniotic tissue component is external to the graft, before rehydrating the lyophilized graft.

[0028] FIG. 4 shows exemplary photos illustrating an exemplary lyophilized amniotic tissue laminate graft in which the stromal layer of each amniotic tissue component is internal to the graft and the epithelial layer of each amniotic tissue component is external to the graft, after rehydrating the lyophilized graft.

[0029] FIG. 5 shows exemplary photos illustrating an exemplary lyophilized amniotic tissue laminate graft in which the stromal layer of each amniotic tissue component is external to the graft and the epithelial layer of each amniotic tissue component is internal to the graft, before rehydrating the lyophilized graft.

[0030] FIG. 6 shows exemplary photos illustrating an exemplary lyophilized amniotic tissue laminate graft in which the stromal layer of each amniotic tissue component is external to the graft and the epithelial layer of each amniotic tissue component is internal to the graft, after rehydrating the lyophilized graft.DETAILED DESCRIPTION

[0031] The present disclosure relates to compositions (e.g., wound dressings or grafts comprising placental tissue), methods of processing and / or making the compositions disclosed herein (e.g., methods of lyophilizing placental tissues), and uses of these compositions for the treatment of wounds (e.g., dermal injuries or wounds). The compositions disclosed herein are also useful for the repair of tissue such as soft tissue including, but not limited to tendons, ligaments, cartilage, and other connective tissue. The placental tissue compositions can further comprise a carrier, such as a pharmaceutically acceptable carrier. Surprisingly, the compositions of placental tissue processed by the methods disclosed herein can provide unexpectedly greater beneficial effects for wound healing as compared to commercially available wound dressing products. Thus, the placental tissue compositions described in this disclosure, including the compositions of placental tissue processed by the methods disclosed herein, may provide more favorable conditions to support the wound healing process versus existing wound dressing products. The placenta has long been considered a disposable and discardable material with little or no value and therefore, may be obtained at little or no cost. Thus, using placental tissue as a component of the compositions of the present disclosure provides for a cost-effective product which may be economical to produce.

[0032] In one aspect, the methods disclosed herein may comprise forming a placental tissue laminate graft by folding or layering placental tissues such that the same first layers of the placental tissue are internal to the graft and the same second layers of the placental tissue are external to each side of the graft once the placental tissues are folded or layered together. The resulting placental tissue graft is thicker with increased adherence between the layers for improved handling by medical practitioners, and the graft can be reversibly applied by medical practitioners at wound and tissue injury sites without side-specific placement requirements. The placental tissue laminate graft can be preserved by, e.g., lyophilization or air-drying, and the method used to preserve the placental tissue laminate product can affect the structural and functional properties, e.g., tissue porosity and / or surface roughness, of the resulting placental tissue product to provide optimized placental tissue products useful for various wound applications. In some aspects, preserving the placental tissue may preserve the structural and mechanical properties of the placental tissue, such as by preventing or reducing delamination of the layers of the placental tissue products, and may provide for an extended shelf-life (e.g., months to years) without ultra-low temperatures for the supply chain. These aspects can beneficially allow more product to be used after manufacturing, thereby reducing manufacturing times and costs.I. EXEMPLARY DEFINITIONS

[0033] In various aspects, the subject of the herein disclosed methods is a vertebrate, e.g., a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. In some aspects, the subject is a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0034] The term “body” as used herein means the body of a subject.

[0035] The term “decellularize” or “decellularization” means removing all or a portion of cellular components and / or contents from tissue, including whole cells, cell membranes, cell organelles, and / or nucleic acids (e.g., DNAs, RNAs, etc.).

[0036] The term “placental tissue” means tissue derived from the placenta in the broadest sense of the word. Placental tissue can be a whole placenta or any portion thereof. “Portions of the placenta” is meant to include chorion, amnion, a chorion and amniotic membrane (e.g., amnio-chorion), Wharton’s jelly, umbilical cord, placental cotyledons, or any combinationsthereof. The placental tissue may be dissected or digested (or combinations thereof) to remove portions, membrane, or structures.

[0037] Compositions comprising placental tissue may include placental tissue that is cryopreserved or dehydrated (e.g., lyophilized or air-dried) or placental tissue that has been previously cryopreserved or dehydrated (e.g., lyophilized or air-dried). Previously cryopreserved or dehydrated (e.g., lyophilized or air-dried) placental tissue refers to placental tissue that has been cryopreserved or dehydrated (e.g., lyophilized or air-dried) and since removed from cryopreservation or dehydration (e.g., lyophilization or air-drying). In some instances, removed from cryopreservation or dehydration (e.g., lyophilization or air- drying)means the placental tissue has been removed from a cryopreservation or dehydration (e.g., lyophilization or air-drying) solution. In some instances, removed from cryopreservation or dehydration (e.g., lyophilization or air-drying) means the placental tissue has been thawed after cryopreservation or rehydrated after dehydration (e.g., lyophilization or air-drying).

[0038] The term “placental cells” means any cell that can be obtained from a placenta, without regard to genetic origin (e.g., maternal vs. fetal), developmental origin (e.g., endodermal, ectodermal, or mesodermal), or differentiation. Placental cells may comprise any placental cells known in the art, for example, mesenchymal stem cells (MSCs), endometrial stromal cells (ESCs), placenta-derived mesenchymal progenitor cells, placental mesenchymal stem cells, fibroblasts, epithelial cells, placental mesenchymal cells, macrophages, and the like. “Placental cells” are further meant to require some feature of live cells such as one or more of metabolic activity, structural integrity (e.g., exclusion of a viability stain such as methylene blue), mitotic activity, signal transduction, and the like.

[0039] The term “placental factor” means any product that is obtainable from a placental tissue (or placental cells). The product may be an angiogenic factor, chemokine, cytokine, growth factor, protease, protease inhibitor, or matrix component.

[0040] The terms “umbilical cord” or “UC” mean umbilical cord that connects a developing fetus to a placenta in a placental mammal. The umbilical cord includes veins and arteries embedded in Wharton's jelly which, in turn, is encased in a layer of amniotic epithelial lining. The Wharton’s jelly and the layer of amniotic epithelial lining comprise the umbilical tissue, while the veins and arteries are the umbilical blood vessels. The native components of umbilical tissue include endogenous cells, extracellular matrix (ECM), and bioactive factors. Endogenous cells found in umbilical tissue include amniotic epithelial cells and stromal cells in Wharton's jelly, such as neonatal fibroblasts, myofibroblasts, mesenchymal stem cells, and macrophages. The ECM of umbilical tissue is largely made up of collagen,mucopolysaccharides (e.g., hyaluronic acid (HA) and chondroitin sulfate). Bioactive factors include, but are not limited to, growth factors, cytokines, and anti-microbial peptides. The native components of umbilical tissue, including endogenous cells, ECM, and bioactive factors, are known to be beneficial for tissue repair and reconstruction.

[0041] The terms “umbilical tissue,” “umbilical cord tissue,” or “UT” refer to the tissue present in the umbilical cord devoid of vessel structure. Umbilical tissue is a rich source of mesenchymal stem cells (MSCs). Umbilical tissue comprises a Wharton’s jelly layer (mesodermal connective tissue) and an amniotic epithelial layer.

[0042] The term “umbilical tissue factors” refers to any factor that originates from umbilical tissue. Examples of umbilical tissue factors may include, but are not limited to, growth factors, cytokines, antimicrobial peptides, proteases and their inhibitors.

[0043] The term “chorionic tissue” or “chorionic membrane” means the chorion or a portion thereof, e.g., trophoblasts, the somatic mesoderm (including the basement membrane and reticular layer), or combinations thereof.

[0044] The term “amniotic tissue” or “amniotic membrane” means the amnion or a portion thereof, e.g., the epithelium, basement membrane, or stroma, which further consists of three contiguous but distinct layers: the inner compact layer, middle fibroblast layer and the outermost spongy layer, or combinations thereof.

[0045] “Native cells” means cells that are native, resident, or endogenous to the tissue sample, i.e., cells that are not exogenously added to the tissue sample.

[0046] “Native factors” means factors that are native, resident, or endogenous to the tissue sample, i.e., factors that are not exogenously added to the tissue sample.

[0047] “Therapeutic cells” as used herein means viable cells native to a given tissue that have retained their native biological functions to dynamically respond to a local microenvironment, for example an injury site or wound. Examples of therapeutic cells include, but are not limited to, fibroblasts, epithelial cells, MSCs, and other tissue-specific cell types, such as osteoblasts or osteoclasts for bone, or CD34+ follicular cells of the skin epidermis, or chondrocytes of hyaline cartilage, or fibrochondrocytes of meniscus, or annulus fibrosus or nucleus pulposus cells of the intervertebral disc, or supportive cell types surrounding peripheral nerve.

[0048] “Therapeutic factors” means tissue-derived factors that promote wound healing or tissue regeneration. For example, placenta- or chorionic membrane-derived factors that promote wound healing or tissue regeneration. Examples include, but are not limited to IGFBPI, adiponectin, a2-macroglobulin, and bFGF. Other examples include, but are notlimited to MMP-9 and TIMP1. Other therapeutic factors include, but are not limited to, TGF- beta 1, beta 2, or beta 3, HGF, VEGF, IGF-1, and BMPs.

[0049] “Substantially free” means present in only a negligible amount or not present at all. For example, when a cell is abundant less than about 20% or less than about 10% or less than about 1% of the amount in an unprocessed sample.

[0050] “Substantial amount” of an element of the present invention, e.g., native factors, therapeutic factors, or selective depletion, means a value at least about 2% or at least 10% in comparison to an unprocessed, fresh tissue sample. A substantial amount can optionally be at least about 50%.

[0051] The term “tissue injury” means an injury of any tissue such as skin or the outer layer of any organ. By injury, it is meant a pathology that involves or results from a mechanical, metabolic, or other insult. Examples of such tissue injuries are bums, wounds, ulcerations, and lacerations, ablations (including laser, freezing, cryo-surgery, heat and electrical ablations), and surgical incisions.

[0052] The term “wound” as used herein means tendon repair, cartilage repair (e.g., femoral condyle, tibial plateau), ACL replacement at the tunnel / bone interface, dental tissue augmentation, fistulas (e.g., Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g., nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g., partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g., diabetic ulcers (e.g., foot), venous leg ulcers), surgical wounds, hernia repair, tendon repair, bladder repair, periosteum replacement, keloids, organ lacerations, epithelial defects, and repair or replacement of a tympanic membrane. Optionally, the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile. Optionally, the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, congenital wound, ulcer, or pressure ulcer. Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure. Optionally, the wound is closed surgically prior to administration. Optionally, the burn is a first-degree burn, second-degree bum (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed bum, or burn wound impetigo.

[0053] The placental products disclosed herein are useful in treating wounds. Non-limiting examples of wound sites to which the placental product can be applied include those that are surgically induced or associated with surgery involving the spine, laminectomy, knee,shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, angiogenesis stimulation, brain / neurological procedures, burn and wound care, and ophthalmic procedures. Direction for such procedures, including the selection of wound sites and / or methodologies, can be found, for example, in WO 2009 / 132186 and US 2010 / 0098743, which are hereby incorporated by reference.

[0054] The term “pieces” as used herein with respect to placental tissue means tissue which has been subject to a disruption process, such as shearing, mincing, dicing, chopping, cutting, homogenizing, macerating, or crushing, in which small individual pieces of tissue are formed. Placental pieces may further be dehydrated, dried, and / or lyophilized. The disruption process may occur before or after dehydration, evaporative air-drying, or lyophilization. The pieces may be uniform or irregular in size.

[0055] The term “particulate” as used herein means individual particles. The particles can be uniform or irregular in size. The particle size can be that described below in this specification.

[0056] The term “sheet” as used herein means a product produced from placental tissues, which may have undergone cutting, decellularization, layer separation, and / or lyophilization.

[0057] The term “foam” as used herein means a product produced from placental tissues, which may have undergone cutting, decellularization, layer separation, and / or lyophilization and which is ground to make particulates, and then, lyophilized after mixing with water at a volume ratio (e.g., a 1:1 volume ratio). A foam type product can comprise a porous structure that looks similar to a sponge.

[0058] The term “immunoprivileged” as used herein means a relatively high resistance against promoting an immune response.

[0059] The term “non-immunogenic” as used herein means not promoting an immune response.

[0060] The terms “room temperature” or “RT” or “ambient temperature” as used herein mean a temperature of 20°-25°C.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0062] The terms “optional” or “optionally” as used herein mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0063] The terms “about” or “approximately” as used herein are defined as being close to as understood by one of skill in the art, and in one non-limiting aspect the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5% of an associated disclosed value. The terms may be removed from the associated disclosed value and the exact value may be used instead.

[0064] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated aspect that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0065] When the lower limit value of a given percentage range does not include the % symbol and / or the percentage type (e.g., w / w, v / v, etc.), then the percentage type for the lower limit value is the same as for the upper limit value of the given percentage range. For example, the percentage range of “0.01 to 0.5% w / w” means “0.01% w / w to 0.5% w / w.”

[0066] The use of the word “a” or “an” when used in conjunction with the terms “comprising”, “having”, “including”, or “containing” (or any variations of these words) may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0067] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0068] For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e., round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 0.42 can be rounded to 0.4.

[0069] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) as used herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0070] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. With respect to the transitional phrases “consist essentially of’ or “consisting essentially of,” in one non-limiting aspect, a basic and novel characteristic of the compositions and methods of the present disclosure are their abilities to treat wounds and / or repair tissue in a subject with composition or dehydrated compositions comprising placental tissue, preferably with compositions comprising dehydrated placental tissue.II. OBTAINING A TISSUE SAMPLE

[0071] In some aspects, obtaining a tissue sample can be performed by those methods known in the art. The method of obtaining a placental tissue sample can depend on the type of tissue sample being obtained. For example, obtaining a placental tissue can occur at the time of childbirth.

[0072] In some aspects, a tissue sample can be, but is not limited to, a whole placenta or portion of a placenta. Portions of the placenta may include chorion, amnion, a chorion and amniotic membrane (e.g., amnio-chorion), Wharton’s jelly, umbilical cord, placental cotyledons or combinations thereof. Accordingly, in some aspects, a placenta or placental tissue can be amniotic tissue, chorionic tissue, umbilical cord tissue, or any combinationthereof. The placental tissue may be dissected or digested (or combinations thereof) to remove portions, membrane, or structures. In some aspects, placental tissue is used in a purposeful wound dressing product with superior biological properties with respect to supporting the wound healing process by allowing cellular proliferation and migration, vascular ingrowth, and / or the formation of granulation tissues.

[0073] In specific aspects, the tissue sample is any placental tissue, such as but not limited to amnion, chorion, amnion with chorion, double layered amnion, chorion with trophoblast, umbilical cords, and any mixture composed of birth tissues. In some aspects, the umbilical tissue has a Wharton’s jelly layer side and an amniotic epithelial layer side. The disclosed methods may comprise producing umbilical tissue (e.g., by removing blood vessels from the received umbilical cords) and / or rinsing the umbilical tissue. In some aspects, umbilical tissue is devoid of viable blood cells. In some instances, one or more anticoagulants can be used to help remove blood and blood products, for instance ACD-A (anticoagulant Citrate dextrose solution, solution A, USP), heparin, dalteparin sodium, and bivalirudin. In some aspects, umbilical tissue is devoid of blood vessels. In some instances, the disclosed compositions are devoid of the two arteries and one vein typically found in the umbilical cord. In some aspects, disclosed is placental tissue comprising umbilical tissue, wherein the umbilical tissue is between 1 cm2and 350 cm2. Removing the blood vessels from the umbilical cord may allow for larger pieces of umbilical tissue to be used instead of having to cut around the blood vessels.

[0074] In some aspects, the placental tissue comprises viable cells native to the placental tissue. Cells native to the placental tissue refers to cells that are present in naturally occurring placental tissue. In some instances, the viable cells may be mesenchymal stem cells, fibroblasts, epithelial cells, or any combination thereof.

[0075] In some aspects, disclosed are compositions comprising placental tissue, wherein the placental tissue comprises one or more engineered channels or no engineered channels.

[0076] In some aspects, the placental tissue comprises one or more growth factors native to the placental tissue. Growth factors native to the placental tissue refer to growth factors that are present in naturally occurring placental tissue. In some instances, the growth factors may be epidermal growth factor (EGF), human growth factor (HGF), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), TGF-pi, 2, and 3, insulin-like growth factor- 1 (IGF-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, TGF-a, Interleukin 10 (IL-10), Interleukin-1 receptor a (IL-lra), Stromal cell-derived factor-1 (SDF-1), Basic fibroblasts growth factor (bFGF), Neutrophil gelatinase-associated lipocalin (N-Gal), Matrix metalloproteinase 8 (MMP8), Tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2,Angiopoietin 2 (hAng2), thrombospondin 2 (TSP2), Platelet derived growth factor AA (PDGF- AA), PDGF-AB, Placental growth factor (PIGF), Insulin-like growth factor (IGFBP1), IGFBP2, IGFBP3, a2-macroglobulin, Adiponectin (hACRP30), and / or Fibronectin.

[0077] In some aspects, the placental tissue further comprises one or more cytokines native to the placental tissue. Cytokines native to the placental tissue refers to cytokines that are present in naturally occurring placental tissue. In some instances, the one or more cytokines may be stromal cell derived factor- 1 (SDF-1 or CXCL12), IL- 10, and / or IL- Ira.

[0078] In some aspects, the placental tissue is capable of releasing placental factors, such as angiogenic factors, optionally at an increased rate over time compared to native placental tissue. In some instances, the placental tissue releases placental factors, such as angiogenic factors, optionally at an increased rate over time compared to native placental tissue. In some instances, the angiogenic factors may be growth factors. For example, the disclosed compositions may optionally have greater release of growth factors in comparison to growth factor levels released by native placental tissue for the same period of time. In some instances, the growth factors may be EGF, HGF, KGF, bFGF, TGF-pl, 2, and 3, IGF-1, VEGF, VEGF- C, VEGF-D, TGF-a, IL- 10, IL- Ira, SDF-1, bFGF, N-Gal, MMP8, TIMP1, TIMP2, hAng2, TSP2, PDGF-AA, PDGF-AB, PIGF, IGFBP1, IGFBP2, IGFBP3, a2-macroglobulin, hACRP30, and / or Fibronectin.

[0079] In specific aspects, the placental tissue sample is chorionic membrane and / or amniotic membrane. Optionally, a layer of epithelial cells of the amniotic membrane may be retained. Optionally, the chorionic membrane or portion thereof may be removed. Optionally, trophoblasts may be removed from the chorionic membrane while retaining the stromal cell layer, reticular layer, and / or basement membrane of the chorionic membrane. The chorionic membrane and / or amniotic membrane optionally secrete factors that stimulate cell migration and / or wound healing. Examples include IGFBP1, adiponectin, a2-macroglobulin, and / or bFGF. Other examples include MMP-9 and TIMP1.

[0080] In some aspects, the chorionic membrane and / or amniotic membrane tissue comprises viable cells native to the chorionic membrane and / or amniotic membrane tissue. Cells native to the chorionic membrane and / or amniotic membrane tissue refers to cells that are present in naturally occurring chorionic membrane and / or amniotic membrane tissue. In some instances, the viable cells may be stromal cells, mesenchymal stem cells, fibroblasts, epithelial cells, or any combination thereof.

[0081] In some aspects, the chorionic membrane and / or amniotic membrane can be immunocompatible. Immunocompatability can be accomplished by any selective depletionstep that removes immunogenic cells or factors or immunogenicity from the placenta tissue. In one aspect, the placental tissue is made immunocompatible by selectively depleting it of functional immunogenic cells. A placenta can be made immunocompatible by selectively removing immunogenic cells from the placenta (or amniotic membrane thereof) relative to therapeutic cells. For example, immunogenic cells can be removed by killing the immunogenic cells or by purification of the placenta therefrom. In some aspects, the placental tissue is made immunocompatible by selectively depleting trophoblasts, for example, by removal of the trophoblast layer. In one aspect, the placenta is made immunocompatible by selective depletion of functional CD 14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay. In some aspects, the placenta is made immunocompatible by selective depletion of vascularized tissue-derived cells. In some aspects, vascularized tissue is removed from the placenta, for example, by lysing red blood cells, by removing blood clots, or a combination thereof. In some aspects, the placenta is made immunocompatible by selective depletion of functional CD 14+ macrophages, trophoblasts, and vascularized tissue-derived cells.

[0082] In some aspects, immunocompatability (or selective depletion) is accomplished by removal or depletion of trophoblasts from the placental product. Trophoblasts can be removed by removing the chorionic membrane from the placental product or by removing trophoblasts from the chorionic membrane while retaining at least one of the basement layer, reticular layer, or stromal cell layer of the chorionic membrane. Such a placental product has one or more of the following superior features: a. is substantially non-immunogenic; b. provides remarkable healing time; and c. provides enhanced therapeutic efficacy.

[0083] In some aspects, trophoblasts are removed while retaining the basement layer, reticular layer, and / or stromal cell layer of the chorionic membrane. Trophoblasts can be removed in any suitable manner which substantially diminishes the trophoblast content of the placental product. Optionally, the trophoblasts are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the chorionic membrane (e.g., MSCs, placental factors, etc.). Optionally, a majority (e.g., substantially all) of the trophoblasts are removed.

[0084] One method of removing trophoblasts comprises treating the placenta (e.g., chorion, amnion, or amino-chorion) with a digestive enzyme such as dispase (e.g., dispase II) and separating the trophoblasts from the placenta. Optionally, the step of separating comprises mechanical separation such as peeling or scraping. Optionally, scraping comprises scrapingwith a soft instrument such as a finger. In some aspects, preparation of birth tissues does not comprise use of a dispase digestive enzyme.

[0085] One method of removing trophoblasts comprises treating the chorionic membrane with dispase for about 30 to about 45 minutes separating the trophoblasts from the placenta. Optionally, the dispase is provided in a solution of about less than about 1% (e.g., about 0.5%). Optionally, the step of separating comprises mechanical separation such as peeling or scraping. Optionally, scraping comprises scraping with a soft instrument such as a finger.

[0086] Useful methods of removing trophoblasts from a placenta (e.g., chorion) are described by Portmann-Lanz et al. (“Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration”; American Journal of Obstetrics and Gynecology (2006) 194, 664-73), (“Isolation and characterization of mesenchymal cells from human fetal membranes”; Journal Of Tissue Engineering And Regenerative Medicine 2007; 1: 296-305.), and (Concise Review: Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells”). In some aspects, trophoblasts are removed before cryopreservation or dehydration (e.g., ly opreservation or air-drying).

[0087] In some aspects, functional macrophages are depleted or removed from the placental tissue. Such a placental product has one or more of the following superior features: a. is substantially non-immunogenic; b. provides remarkable healing time; and c. provides enhanced therapeutic efficacy.

[0088] Functional macrophages can be removed in any suitable manner which substantially diminishes the macrophage content of the placental product. Optionally, the macrophages are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g., MSCs, placental factors, etc.). Optionally, a majority (e.g., substantially all) of the macrophages are removed.

[0089] One method of removing immune cells such as macrophages comprises killing the immune cells by rapid freezing rates such as 60-100 °C / min. Although immune cells can be eliminated by rapid freezing rates, such a method can also be detrimental to therapeutic cells such as stromal cells (e.g., MSCs). CD 14+ macrophages can be selectively killed by refrigerating the placenta for a period of time (e.g., for at least about 10 min such as for about 30-60 mins) at a temperature above freezing (e.g., incubating at 2-8 °C) and then freezing the placenta (e.g., incubating at -80 °C. +5 °C). Optionally, the step of freezing comprises freezing at a rate of less than 10° / min (e.g., less than about 5° / min such as at about l° / min).

[0090] In some aspects, the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g., containing DMSO) for a period of time sufficient to allow the cryopreservation medium to penetrate (e.g., equilibrate with) the placental tissues. Optionally, the step of freezing comprises reducing the temperature at a rate of about 1 min. Optionally, the step of freezing comprises freezing at a rate of less than 10 min (e.g., less than about 5 min such as at about 1 min).

[0091] In some aspects, the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g., containing DMSO) at a temperature of about -10-15 °C (e.g., at 2-8 °C) for at least about any of: 10 min, 20 min, 30 min, 40 min, or 50 min. In another aspect the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g., containing DMSO) at a temperature of about -10-15 °C (e.g., at 2-8 °C) for about any of: 10-120, 20-90 min, or 30-60 min. Optionally, the step of freezing comprises freezing at a rate of less than 10 min (e.g., less than about 57min such as at about 17min).

[0092] In some aspects, vascularized tissue-derived cells (or vascularized tissue) are depleted or removed from the placental tissue. Such a placental product has one or more of the following superior features: a. is substantially non-immunogenic; b. provides remarkable healing time; and c. provides enhanced therapeutic efficacy.

[0093] Vascularized tissue-derived cells can be removed in any suitable manner which substantially diminishes such cell content of the placental product. Optionally, the vascularized tissue-derived cells are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g., MSCs, placental factors, etc.). In some aspects, removal of vascularized tissue-derived cells comprises rinsing the amniotic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells. In some aspects, removal of vascularized tissue-derived cells comprises treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution). In some aspects, removal of vascularized tissue-derived cells comprises rinsing the amniotic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution).

[0094] In some aspects, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord. In some aspects, the chorion on the umbilical side of the placenta is not removed due to the vascularization on this side. In some aspects, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on theside opposite of the umbilical cord and rinsing the amniotic membrane and chorionic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells. In some aspects, the chorionic membrane is retained and removal of vascularized tissue- derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and treating the amniotic membrane and chorionic membrane with an anticoagulant (e.g., citrate dextrose solution). In some aspects, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord, rinsing the chorionic membrane amniotic membrane (e.g., with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g., citrate dextrose solution).

[0095] In some aspects, the placental tissue is selectively depleted of immunogenicity as demonstrated by a reduction in LPS stimulated TNF-a release. In some aspects, the placental product is selectively depleted of macrophages. In some aspects, TNF-a is depleted by killing or removal of macrophages. In some aspects, TNF-a is functionally depleted by treatment with IL- 10, which suppresses TNF-a secretion.

[0096] In some aspects, a placental tissue sample does not comprise cultured cells. For example, the cells present in the tissue sample would be considered native to the tissue sample and non-cultured if the native cells have not previously been removed from the tissue sample and plated, seeded, cultured or in any other way allowed to adhere to a plastic or protein surface for any amount of time. Cells that have been previously removed from the tissue sample and plated, seeded, cultured or in any other way allowed to adhere to a plastic or protein surface for any amount of time are referred to herein as “cultured cells.”

[0097] In some aspects, a tissue sample can be cut to a desired size. Cutting a tissue sample to a desired size can occur prior to freezing or dehydrating the tissue sample (i.e., before or after contacting the tissue sample with a cyroprotectant or lyoprotectant solution). In some aspects, a tissue sample can be minced. Mincing a tissue sample can occur prior to freezing or dehydrating the tissue sample (i.e., before or after contacting the tissue sample with a cyroprotectant or lyoprotectant solution).

[0098] In some aspects, a tissue sample can be decellularized and / or disinfected (e.g., viral inactivation) placental tissue (e.g., amnion, chorion, amnion with chorion, double layered amnion, chorion with trophoblast, umbilical cords, and any mixture composed of birth tissues). In some aspects, a tissue sample can be treated with an antibiotic. In some aspects, a tissue sample can be treated with an antibiotic prior to freezing or dehydrating (e.g., before or aftercontacting the tissue sample with a cyroprotectant or lyoprotectant solution). In some aspects, the disclosed methods may comprise obtaining or receiving placental tissue (e.g., amnion, chorion, amnion with chorion, double layered amnion, chorion with trophoblast, umbilical cords, and any mixture composed of birth tissues). In some aspects, the placental tissue may be fresh or previously cryopreserved or dehydrated (e.g., lyophilized or air-dried). In some aspects, impacts to the placental tissue’s natural tissue characteristics, such as extracellular matrix and mechanical properties, may be minimized such that one or more of the placental tissue’s natural tissue characteristics, such as extracellular matrix and mechanical properties, are preserved. In some aspects, cellular components, microorganisms, and / or viral particles may be removed, washed away, or inactivated, thus promoting additional safety features to placental (e.g., amnion, chorion, amnion with chorion, double layered amnion, chorion with trophoblast, umbilical cords, and any mixture composed of birth tissues) tissue-based devices.III. METHODS OF CRYOPRESERVING

[0099] In some aspects, compositions and methods of the present disclosure comprise placental tissue that has been cryopreserved for a period of time. A placental product may be cryopreserved by incubation at freezing temperatures (e.g., at -80 °C ± 5 °C, or at -40 °C ± 5 °C, or at 0 °C ± 5 °C, etc.) in a cryopreservative medium. In some aspects, cryopreservation is transient and is part of a freeze drying cycle (e.g., a lyophilization cycle).

[0100] Cryopreservation can comprise, for example, incubating the placental product at 4 °C for 30-60 min, and then incubating at -80 °C until use. The placental product may then be thawed for use. Optionally, the placental product is cryopreserved in a manner such that cell viability is retained surprisingly well after a freeze-thaw cycle.

[0101] In some aspects, cryopreservation comprises storage of a placental tissue in a cry opreservation medium comprising one or more cell-permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof. In some aspects, cryopreservation does not comprise storage of a placental tissue in a cryopreservation media that comprises one or more cell-permeating cryopreservatives and / or one or more non cellpermeating cryopreservatives. Optionally, the cry opreservation medium comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof. In some aspects, the cryopreservation medium does not comprise one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof. Optionally, the cryopreservation medium comprises one or more non cell-permeatingcryopreservatives selected from poly vinylpyrrolidone, a hydroxy ethyl starch, a polysaccharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof. In some aspects, the cryopreservation medium does not comprise one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysaccharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof. In some aspects, the cryopreservation medium does not comprise trehalose. Other examples of useful cryopreservatives are described in “Cryopreservation” (BioFiles Volume 5 Number 4-Sigma- Aldrich® datasheet).

[0102] In some aspects, the cry opreservation medium comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO. In some aspects, the cryopreservation medium does not comprise a cell-permeating cryopreservative composed primarily of DMSO. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

[0103] In some aspects, the cryopreservation medium comprises DMSO. Optionally, the cryopreservation medium does not comprise glycerol in a majority amount. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

[0104] In some aspects, the cryopreservation medium comprises additional components such as albumin (e.g., HSA or BSA), an electrolyte solution (e.g., Plasma-Lyte), or a combination thereof.

[0105] In some aspects, the cryopreservation medium comprises 1% to about 15% albumin by weight and about 5% to about 20% cryopreservative by volume (e.g., about 10%). Optionally, the cryopreservative comprises DMSO (e.g., in a majority amount).

[0106] Thawed placental tissue of the disclosed methods can comprise at least 70% viable cells. In some aspects, thawed tissue can comprise at least, at most, exactly, or between any two of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% viable cells. In some aspects, thawed cryopreserved tissue can be decellularized and / or comprise substantially no viable cells. Percent viability of cells after thawing is based on the percent of viable cells that were in the starting tissue sample prior to being cryopreserved.

[0107] In some aspects, cryopreservation comprises placing the placenta on nitrocellulose paper. In some aspects, the placenta is cut into a plurality of sections before cryopreservation. In some aspects, the placenta is not cut into a plurality of sections before cryopreservationand / or dehydration (e.g., lyophilization or air-drying). Optionally, the sections are placed on nitrocellulose paper before refrigeration.

[0108] In some aspects, cryopreservation comprises the layering of one or more placental tissues prior to cryopreservation. In some aspects, cryopreservation comprises the layering of one or more placental tissues after cryopreservation. Layering of one or more placental tissues may be achieved by, e.g., folding or stacking one or more placental tissues.IV. METHODS OF DEHYDRATING

[0109] In some aspects, compositions and methods of the present disclosure comprise placental tissues that may be dehydrated and / or that have previously been dehydrated. Dehydration of the placental tissue may be conducted by any suitable means known to one of skill in the art, including, but not limited to lyophilizing (freeze-drying), evaporative air drying (e.g., heat-drying or thermal drying with heated air under gravity convection or forced air convection conditions; or air-drying under ambient or room temperature conditions under gravity convection or forced air convection conditions), indirect drying (e.g., vacuum drying), contact drying (e.g., drying through contact with a heated material or source), dielectric drying (e.g., use of radiofrequency or microwaves), infrared drying, and / or supercritical drying. The dehydrated placental tissue is considered “dehydrated” or “dry” when it has a water content (moisture content) of less than 15% w / w, or less than 14% w / w, or less than 13% w / w, or less than 12% w / w, or less than 10% w / w, or less than 9% w / w, or less than 8% w / w, or less than 7% w / w, or less than 6% w / w, or less than 5% w / w, or less than 4% w / w, or less than 3% w / w, or less than 2% w / w, less than 1% w / w, or less than 0.5% w / w. In some aspects, the water content is less than 5% w / w, or less than 4% w / w, or less than 3% w / w. The dehydrated placental tissue may, for example, have a water content of 0, 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% w / w, or any percentage derivable therein. The water content (moisture content) of the dehydrated tissue may be determined by methods known to one of skill in the art such as by Karl Fischer titration or by oven drying.

[0110] In one aspect, the dehydrated placental tissue is lyophilized or dried placental tissue. In some aspects, the placental tissue may be viable, meaning that the placental tissue comprises viable cells. The viable cells may be native cells or exogenous cells, or mixtures thereof. In some aspects, the placental tissue may be non-viable, meaning that no viable cells are present in or on the tissue. The placental tissue may be rendered non-viable by any suitable means known to one of skill in the art, including, but not limited to cell lysis, sterilization, irradiation, or cell removal by enzymatic or physical means. In some aspects, the compositions of thepresent disclosure and / or the placental tissue of the present disclosure are sterilized. In some aspects, the placental tissue is dehydrated by lyophilization resulting in lyophilized placental tissue. In some aspects, the placental tissue is rendered non-viable by the methods disclosed herein.A. METHODS OF DRYING

[0111] In some aspects, compositions and methods of the present disclosure comprise placental tissues that may be dried and / or that have previously been. In some aspects, a dried and / or previously dried placental tissue of the present disclosure may be reconstituted to have characteristics similar and / or substantially the same as fresh placental tissues. A placental tissue of the present disclosure may be used fresh or may be preserved by drying for a period of time.

[0112] Disclosed are methods of drying a placental tissue sample comprising obtaining a placental tissue sample, and performing a drying step of the placental tissue sample. The drying step may comprise evaporative air drying (e.g., heat-drying or thermal drying with heated air under gravity convection or forced air convection conditions; or air-drying or drying under ambient or room temperature conditions under gravity convection or forced air convection conditions), indirect drying (e.g., vacuum drying), contact drying (e.g., drying through contact with a heated material or source), dielectric drying (e.g., use of radiofrequency or microwaves), infrared drying, and / or supercritical drying.

[0113] In some aspects, drying the tissue sample can be performed at room temperature. In some aspects, drying the tissue sample can be performed at a temperature range of 15 °C to 50 °C (e.g., at least, at most, exactly, or between any two of 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, or 50 °C). In some aspects, drying the tissue sample comprises heat-drying the tissue sample (e.g., in an oven) and can be performed at a temperature range of 35 °C to 45 °C (e.g., 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C). In some aspects, drying the tissue sample comprises heat-drying the tissue sample, and the final drying temperature is about 40 °C. In some aspects, drying the tissue sample comprises air-drying the tissue sample (e.g., in a laminar flow hood) and can be performed at a temperature range of 15 °C to 25 °C (e.g., 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, or 25 °C). In some aspects, drying the tissue sample comprises air-drying the tissue sample, and the final drying temperature is about 20 °C.

[0114] In some aspects, the sample can be added for purposes of drying the tissue, wherein the tissue can be added prior to achieving the final drying temperature. In some aspects, the step of drying the tissue sample can involve providing steadily increasing temperatures. In suchinstances, the temperature can be increased at a rate between 1 and 20 °C / min (e.g., at least, at most, exactly, or between any two of 1 °C / min, 2 °C / min, 3 °C / min, 4 °C / min, 5 °C / min, 6 °C / min, 7 °C / min, 8 °C / min, 9 °C / min, 10 °C / min, 11 °C / min, 12 °C / min, 13 °C / min, 14 °C / min, 15 °C / min, 16 °C / min, 17 °C / min, 18 °C / min, 19 °C / min, or 20 °C / min). In such instances, the temperature can be increased at a rate of about 10 °C / min. In some instances, steadily increasing the temperature during drying can be used to avoid killing the tissue or native cells contained in the sample.

[0115] In some aspects, the sample can be held at the final drying temperature for a specific amount of time. In some aspects, the sample is held at the final drying temperature for 0.1 minutes to 10 minutes (e.g., at least, at most, exactly, or between any two of 0.1 min, 0.2 min, 0.3 min, 0.4 min, 0.5 min, 0.6 min, 0.7 min, 0.8 min, 0.9 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min). In some aspects, the sample is held at the final drying temperature for about 2 minutes.

[0116] In some aspects, drying the tissue sample can be performed at a humidity of 0.1% to 10% (e.g., at least, at most, exactly, or between any two of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%). In some aspects, drying the tissue sample can be performed at a humidity of about 1%.

[0117] In some aspects, drying the tissue sample can be performed at a pressure of 0 mBar to 1200 mBar (e.g., at least, at most, exactly, or between any two of 0 mBar, 20 mBar, 40 mBar, 60 mBar, 80 mBar, 100 mBar, 120 mBar, 140 mBar, 160 mBar, 180 mBar, 200 mBar, 220 mBar, 240 mBar, 260 mBar, 280 mBar, 300 mBar, 320 mBar, 340 mBar, 360 mBar, 380 mBar, 400 mBar, 420 mBar, 440 mBar, 460 mBar, 480 mBar, 500 mBar, 520 mBar, 540 mBar, 560 mBar, 580 mBar, 600 mBar, 620 mBar, 640 mBar, 660 mBar, 680 mBar, 700 mBar, 720 mBar, 740 mBar, 760 mBar, 780 mBar, 800 mBar, 820 mBar, 840 mBar, 860 mBar, 880 mBar, 900 mBar, 920 mBar, 940 mBar, 960 mBar, 980 mBar, 1000 mBar, 1020 mBar, 1040 mBar, 1060 mBar, 1080 mBar, 1100 mBar, 1120 mBar, 1140 mBar, 1160 mBar, 1180 mBar, or 1200 mBar). In some aspects, drying the tissue sample can be performed at atmospheric pressure, e.g., about 1013.25 mBar.

[0118] Also disclosed are methods of preparing a placental tissue sample comprising obtaining a placental tissue sample, drying the tissue sample, and further comprising a step of reconstituting the dried placental tissue. In some aspects, reconstituted dried tissue can be decellularized and / or comprise substantially no viable cells.

[0119] In some aspects, after dehydrating the tissue, the tissue can be cut to a desired size and / or shape. In some aspects, prior to dehydrating the tissue, the tissue is cut to a desired size and / or shape.

[0120] In some aspects, dehydration comprises the layering of one or more placental tissues prior to dehydration. In some aspects, dehydration comprises the layering of one or more placental tissues after dehydration. Layering of one or more placental tissues may be achieved by, e.g., folding or stacking one or more placental tissues.B. METHODS OF LYOPHILIZING

[0121] In some aspects, compositions and methods of the present disclosure comprise placental tissues that may be lyophilized and / or that have previously been lyophilized. In some aspects, a lyophilized and / or previously lyophilized placental tissue of the present disclosure may be reconstituted to have characteristics similar and / or substantially the same as fresh placental tissues. In some aspects, disclosed herein are methods of lyophilizing a placental tissue sample comprising obtaining a placental tissue sample, optionally contacting the placental tissue sample with a lyoprotectant solution with the caveat that the lyoprotectant solution does not comprise trehalose, freezing the placental tissue sample in one or more steps, performing a first drying step of the placental tissue sample after freezing, and performing at least a second drying step of the placental tissue sample after the first drying step.

[0122] In some aspects, disclosed herein are methods of lyophilizing a placental tissue sample comprising obtaining a placental tissue sample, optionally contacting the placental tissue sample with a lyoprotectant solution with the caveat that the lyoprotectant solution does not comprise trehalose, freezing the placental tissue sample in one or more steps, performing a first drying step of the placental tissue sample after freezing, performing a second drying step of the placental tissue sample after the first drying step, and performing at least a third drying step after the second drying step.

[0123] In some aspects, disclosed herein are methods of lyophilizing a placental tissue sample comprising obtaining a placental tissue sample, optionally contacting the placental tissue sample with a lyoprotectant solution with the caveat that the lyoprotectant solution does not comprise trehalose, freezing the placental tissue sample in one or more steps, performing a first drying step of the placental tissue sample after freezing, performing a second drying step of the placental tissue sample after the first drying step, performing a third drying step after the second drying step, and performing at least a fourth drying step after the third drying step.

[0124] In some aspects, disclosed herein are methods of lyophilizing a placental tissue sample comprising obtaining a placental tissue sample, optionally contacting the placentaltissue sample with a lyoprotectant solution with the caveat that the lyoprotectant solution does not comprise trehalose, vacuum pre-sealing the placental sample, freezing the placental tissue sample in two or more steps, evacuating substantially all air from the placental sample to create a vacuum, performing a first drying step of the placental tissue sample after freezing, performing a second drying step of the placental tissue sample after the first drying step, performing a third drying step after the second drying step, performing a fourth drying step after the third drying step, and releasing the vacuum.

[0125] In some aspects, disclosed herein are methods of lyophilizing a placental tissue sample comprising obtaining a placental tissue sample, optionally contacting the placental tissue sample with a lyoprotectant solution with the caveat that the lyoprotectant solution does not comprise trehalose, vacuum pre-sealing the placental sample, freezing the placental tissue sample by reducing the temperature from an ambient temperature (e.g., about 25 °C) to about or exactly 0 °C to -40 °C (e.g., to at least, at most, exactly, between any two of, or about 0 °C,-1 °C, -2 °C, -3 °C, -4 °C, -5 °C, -6 °C, -7 °C, -8 °C, -9 °C, -10 °C, -11 °C, -12 °C, -13 °C, -14°C, -27 °C, -28 °C, -29 °C, -30 °C, -31 °C, -32 °C, -33 °C, -34 °C, -35 °C, -36 °C, -37 °C, -38°C, -39 °C, or -40 °C), maintaining the placental tissue sample at a freeze temperature of about or exactly -25 °C to - 40 °C (e.g., at least, at most, exactly, between any two of, or about -25°C, -38 °C, -39 °C, or -40 °C), evacuating substantially all air from the placental sample to create a vacuum, performing a first drying step of the placental tissue sample after freezing comprising raising temperature of the sample from about or exactly -25 °C to - 40 °C (e.g., at least, at most, exactly, between any two of, or about -25 °C, -26 °C, -27 °C, -28 °C, -29 °C, - 30 °C, -31 °C, -32 °C, -33 °C, -34 °C, -35 °C, -36 °C, -37 °C, -38 °C, -39 °C, or -40 °C) to between about or exactly -2 °C to 2 °C (e.g., -2 °C, -1 °C, 0 °C, 1 °C, or 2 °C), performing a second drying step of the placental tissue sample after the first drying step comprising raising temperature of the sample from about or exactly -2 °C to 2 °C (e.g., at least, at most, exactly, between any two of, or about -2 °C, -1 °C, 0 °C, 1 °C, or 2 °C) to between about or exactly 14 °C to 16 °C (e.g., at least, at most, exactly, between any two of, or about 14 °C, 15 °C, or 16 °C), performing a third drying step after the second drying step comprising raising temperature of the sample from about or exactly 14 °C to 16 °C (e.g., at least, at most, exactly, between any two of, or about 14 °C, 15 °C, or 16 °C) to between about or exactly 23 °C to 27 °C (e.g., at least, at most, exactly, between any two of, or about 23 °C, 24 °C, 25 °C, 26 °C, or 27 °C), and performing a fourth drying step after the third drying step comprising maintaining thetemperature at about or exactly 23 °C to 27 °C (e.g., at least, at most, exactly, between any two of, or about 23 °C, 24 °C, 25 °C, 26 °C, or 27 °C), and releasing vacuum.

[0126] Also disclosed are methods of preparing a placental tissue sample comprising obtaining a placental tissue sample, performing a lyophilization cycle on the placental tissue sample with the caveat that the placental tissue sample was not contacted with a lyoprotectant solution that comprised trehalose, and further comprising a step of reconstituting the lyophilized placental tissue. In some aspects, reconstituted lyophilized tissue can be decellularized and / or comprise substantially no viable cells.

[0127] In some aspects, after reconstituting the lyophilized placental tissue, the tissue can then be cut to a desired size. In some aspects, the placental tissue is cut to a desired size prior to lyophilization.

[0128] In some aspects, after reconstituting the lyophilized placental tissue, the tissue can then be cut to a desired size. In some aspects, the placental tissue is cut to a desired size prior to lyophilization.

[0129] In some aspects, lyophilization comprises the layering of one or more placental tissues prior to lyophilization. In some aspects, lyophilization comprises the layering of one or more placental tissues after lyophilization. Layering of one or more placental tissues may be achieved by, e.g., folding or stacking one or more placental tissues. a. Contacting The Tissue Sample With A Lyoprotectant Solution

[0130] In some aspects, placental tissue samples are optionally contacted with a lyoprotectant solution prior to lyophilization. In some aspects, the lyoprotectant solution does not comprise trehalose. In some aspects, contacting the tissue sample with a lyoprotectant solution can include one or more short and / or prolonged contacts. For example, in some aspects, a tissue sample can be exposed to or contacted with a lyoprotectant solution for at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some aspects, the tissue sample can be exposed or contacted to a lyoprotectant solution for at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some aspects, the tissue sample can be exposed or contacted to a lyoprotectant solution for at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 14, 21 days. In some aspects, the tissue sample can be exposed or contacted to a lyoprotectant solution for at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, placental tissues are not contacted with a lyoprotectant solution prior to lyophilization.

[0131] In some aspects, contacting the tissue sample with a lyoprotectant solution can be the same as exposing the tissue sample to a lyoprotectant solution or soaking the tissue sample in a lyoprotectant solution.

[0132] As described herein, a lyoprotectant solution comprises at least one lyoprotectant. As described herein, a lyoprotectant solution does not comprise the lyoprotectant trehalose. In some aspects, a lyoprotectant can include but is not limited to polyhydroxy compounds such as sugars, polyalcohols, raffinose, and other non-reducing polysaccharides, and their derivatives. In some aspects, any one or more of the foregoing polyhydroxy compounds may be expressly excluded from a lyoprotectant solution utilized according to the compositions and methods disclosed herein.

[0133] In some aspects, the lyoprotectant solution can comprise one or more antioxidants. In some aspects, the one or more antioxidants can be epigallocatechin gallate (EGCG) or catechin. In some aspects, an antioxidant can be ascorbic acid, L-camosine, spermine, phloretine, a-tocopherol, P-carotene, coenzyme Q10, lutein, melatonin, butylated hydroxytoluene, y-tocopherol, lutein, N-acetyl-L-cysteine, mitoquinone, hydroquinone, lipoic acid, glutathione, carotenoids, polyphenols, retinol, and / or tocotrienol. In some aspects, any one or more of the foregoing antioxidants may be expressly excluded from a lyoprotectant solution utilized according to the compositions and methods disclosed herein.

[0134] In some aspects, lyoprotectant solution can comprise saline, DMSO, antibiotics, bulking agents, excipients, or a combination thereof. In some aspects, a lyoprotectant can comprise other reagents that can improve lyophilization performance. Any one or more of the foregoing components may be expressly excluded from a lyoprotectant solution utilized according to the compositions and methods disclosed herein.

[0135] In some aspects, concentration of a lyoprotectant and / or antioxidants present in a lyoprotectant solution and the length of time for contacting a tissue sample with a lyoprotectant solution can be dependent on the type and size of the tissue sample. Based on the teachings herein, one of skill in the art using routine methods would understand how to adjust the concentrations and contacting times.

[0136] In some aspects, contacting the tissue sample with a lyoprotectant solution can occur at temperatures between 0 °C and 3 °C. In some aspects, contacting a tissue sample with a lyoprotectant solution can occur at about 4 °C. In some aspects, contacting a tissue sample with a lyoprotectant solution comprises a first step in freezing the tissue sample. In some aspects, contacting a tissue sample with a lyoprotectant solution can occur at about 25 °C. . In some aspects, contacting a tissue sample with a lyoprotectant solution can occur over a periodof time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30 minutes. b. Freezing The Tissue Sample

[0137] In some aspects, freezing the tissue sample can be performed at a temperature range(e.g., a freezing temperature) of -80 °C to -4 °C (e.g., at least, at most, exactly, or between any two of -80 °C, -79 °C, -78 °C, -77 °C, -76 °C, -75 °C, -74 °C, -73 °C, -72 °C, -71 °C, -70°C, -69 °C, -68 °C, -67 °C, -66 °C, -65 °C, -64 °C, -63 °C, -62 °C, -61 °C, -60 °C, -59 °C, -58°C, -57 °C, -56 °C, -55 °C, -54 °C, -53 °C, -52 °C, -51 °C, -50 °C, -49 °C, -48 °C, -47 °C, -46°C, -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C, -38 °C, -37 °C, -36 °C, -35 °C, -34°C, -33 °C, -32 °C, -31 °C, -30 °C, -29 °C, -28 °C, -27 °C, -26 °C, -25 °C, -24 °C, -23 °C, -22°C, -21 °C, -20 °C, -19 °C, -18 °C, -17 °C, -16 °C, -15 °C, -14 °C, -13 °C, -12 °C, -11 °C, -10°C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, or -4 °C). In some aspects, freezing the tissue sample can be performed at a temperature range (e.g., a freezing temperature) of -70 °C to -4 °C. In some aspects, freezing the tissue sample can be performed at a temperature range (e.g., a freezing temperature) of -50 °C to -4 °C. In some aspects, freezing the tissue sample can be performed at a temperature range (e.g., a freezing temperature) of -45 °C to -20 °C. In some aspects, freezing the tissue sample can be performed at a temperature range (e.g., a freezing temperature) of -45 °C to -35 °C. In some aspects, freezing the tissue sample can be performed at a freezing temperature of about -40 °C. Any one or more of the foregoing freezing temperatures may be expressly excluded from the methods described herein.

[0138] In some aspects, the sample can be added for purposes of freezing the tissue, wherein the tissue can be added prior to achieving the final freezing temperature. In some aspects, the step of freezing the tissue sample can involve avoiding a flash freeze and instead providing a steady cooling to freezing temperatures. In such instances, the temperature can be decreased or ramped down at a rate between 0.1 and 10 °C / min (e.g., at least, at most, exactly, or between any two of 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1 °C / min, 1.1 °C / min, 1.2 °C / min, 1.3 °C / min, 1.4 °C / min,1.5 °C / min, 1.6 °C / min, 1.7 °C / min, 1.8 °C / min, 1.9 °C / min, 2 °C / min, 2.1 °C / min, 2.2 °C / min,2.3 °C / min, 2.4 °C / min, 2.5 °C / min, 2.6 °C / min, 2.7 °C / min, 2.8 °C / min, 2.9 °C / min, 3 °C / min, 3.1 °C / min, 3.2 °C / min, 3.3 °C / min, 3.4 °C / min, 3.5 °C / min, 3.6 °C / min, 3.7 °C / min, 3.8 °C / min, 3.9 °C / min, 4 °C / min, 4.1 °C / min, 4.2 °C / min, 4.3 °C / min, 4.4 °C / min, 4.5 °C / min, 4.6 °C / min, 4.7 °C / min, 4.8 °C / min, 4.9 °C / min, 5 °C / min, 5.1 °C / min, 5.2 °C / min, 5.3 °C / min,5.4 °C / min, 5.5 °C / min, 5.6 °C / min, 5.7 °C / min, 5.8 °C / min, 5.9 °C / min, 6 °C / min, 6.1 °C / min,6.2 °C / min, 6.3 °C / min, 6.4 °C / min, 6.5 °C / min, 6.6 °C / min, 6.7 °C / min, 6.8 °C / min, 6.9 °C / min, 7 °C / min, 7.1 °C / min, 7.2 °C / min, 7.3 °C / min, 7.4 °C / min, 7.5 °C / min, 7.6 °C / min, 7.7 °C / min, 7.8 °C / min, 7.9 °C / min, 8 °C / min, 8.1 °C / min, 8.2 °C / min, 8.3 °C / min, 8.4 °C / min, 8.5 °C / min, 8.6 °C / min, 8.7 °C / min, 8.8 °C / min, 8.9 °C / min, 9 °C / min, 9.1 °C / min, 9.2 °C / min,9.3 °C / min, 9.4 °C / min, 9.5 °C / min, 9.6 °C / min, 9.7 °C / min, 9.8 °C / min, 9.9 °C / min, or 10 °C / min). In such instances, the temperature can be decreased at a rate between 0.1 °C / min and 5 °C / min. Any one or more of the foregoing temperature decrease rates may be expressly excluded from the methods described herein. In some instances, flash freezing can cause formation of water crystals that can kill the tissue-resident cells and alter the structure of the tissue matrix. In such instances, a slower freeze can be used to avoid killing the tissue or native cells contained therein.

[0139] In some aspects, following freezing of the tissue sample, the tissue can be stored (e.g., “held”) at a freezing temperature over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100, or greater than 100 minutes. In some aspects, a tissue sample can be stored at a freezing temperature for about 40 minutes to about 80 minutes, about 50 minutes to about 70 minutes, or about 60 minutes. In some aspects, a tissue sample can be stored at a freezing temperature for any period of time, for example but not limited to, at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, or greater than 7, days, weeks, or months. Any one or more of the foregoing times for which the tissue sample is held at a freezing temperature may be expressly excluded from the methods described herein.

[0140] In some aspects, freezing the tissue sample can be performed over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, or greater than 360 minutes.

[0141] In some aspects, freezing a tissue sample can be performed over one or more freezing steps. In some aspects, freezing a tissue sample comprises 1, 2, or greater than 2 freezing steps after contacting the tissue sample with a lyoprotectant solution. In some aspects, a first freezing step after contacting the tissue sample with a lyoprotectant solution can be performed over a period of time, such as over at least, at most, exactly, or between any two ofI, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or greater than 120 minutes, e.g., about 40 minutes to about 80 minutes, about 50 minutes to about 70 minutes, or about 60 minutes. In some aspects, the period of time over which the first freezing step after contacting the tissue sample with a lyoprotectant solution is performed includes (1) the time over which the temperature is decreased or ramped down from the temperature at which the tissue sample is contacted with a lyoprotectant solution to the freezing temperature and / or (2) the time over which the tissue sample is held at the freezing temperature. In some aspects, the period of time over which the first freezing step after contacting the tissue sample with a lyoprotectant solution is performed includes (1) ramping down from the temperature at which the tissue sample is contacted with a lyoprotectant solution to the freezing temperature from at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or greater than 100 minutes, and / or(2) holding the tissue sample at the freezing temperature for at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, or greater than 100 minutes.

[0142] In some aspects, a second freezing step after contacting the tissue sample with a lyoprotectant solution can be performed over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or greater than150 minutes, e.g., about 100 minutes to about 140 minutes, about 110 minutes to about 130 minutes, or about 120 minutes. c. Evacuating The Tissue Sample After Freezing

[0143] In some aspects, following freezing of a tissue sample, an evacuation step is conducted. In some aspects, a vacuum pressure is initiated on the tissue sample. For example, in some aspects, a vacuum SP (mTorr) is at a range of about 0 to about 500 mTorr (e.g., an mTorr range of 0 to 500, e.g., at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 mTorr).

[0144] The period of time over which vacuum pressure is maintained will depend on the specific lyophilization equipment used. For example, in some aspects, the vacuum pressure is maintained over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30 minutes. d. Performing A First Drying Step Of The Tissue Sample After Freezing & Evacuating

[0145] In some aspects, following freezing and evacuation of a tissue sample, a drying step is conducted. In some aspects, the first drying step of the tissue sample after freezing and evacuation occurs at a vacuum SP (mTorr) of about 150 to 250, or about 175 to 225, or about 200 mTorr. Any one or more of the foregoing vacuum SP values may be expressly excluded from the methods described herein.

[0146] In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and -15 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and -10 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and -5 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and 0 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and +15 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and +10 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -45 °C and +5 °C. In some aspects, the first drying step of the tissue sample after freezing occursbetween at least, at most, exactly, or between any two of -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C, -38 °C, -37 °C, -36 °C, -35 °C, -34 °C, -33 °C, -32 °C, -31 °C, -30 °C, -29°C, -28 °C, -27 °C, -26 °C, -25 °C, -24 °C, -23 °C, -22 °C, -21 °C, -20 °C, -19 °C, -18 °C, -17°C, -16 °C, -15 °C, -14 °C, -13 °C, -12 °C, -11 °C, -10 °C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4°C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, or 5 °C. In some aspects, the temperature of the first drying step can be the same as the freezing temperature. In some aspects, the temperature of the first drying step can be at least, at most, exactly, or between any two of 1 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, or 50 °C higher than the temperature of the freezing step. Any one or more of the foregoing first drying step temperatures may be expressly excluded from the methods described herein.

[0147] In some aspects, the first drying step of the tissue sample after freezing occurs between -10 °C and 10 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -5 °C and 5 °C. In some aspects, the first drying step of the tissue sample after freezing occurs between -2 °C and 2 °C. In some aspects, the first drying step of the tissue sample after freezing occurs at about 0 °C. In some aspects, the first drying step of the tissue sample after freezing raises the temperature from the freezing temperature to at least, at most, exactly, or between any two of -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C,-38 °C, -37 °C, -36 °C, -35 °C, -34 °C, -33 °C, -32 °C, -31 °C, -30 °C, -29 °C, -28 °C, -27 °C,-26 °C, -25 °C, -24 °C, -23 °C, -22 °C, -21 °C, -20 °C, -19 °C, -18 °C, -17 °C, -16 °C, -15 °C,-14 °C, -13 °C, -12 °C, -11 °C, -10 °C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, or 5 °C. In some aspects, the first drying step of the tissue sample after freeze comprises raising the temperature from the freezing temperature to between about -5 °C to about 5 °C, between about -2 °C to about 2 °C, or about 0 °C. Any one or more of the foregoing first drying step temperatures may be expressly excluded from the methods described herein.

[0148] In some aspects, the step of drying the tissue sample can involve avoiding a sudden shock temperature increase, and instead comprises providing a steady warming of the tissue from the freezing temperature. In such instances, the temperature can be increased at a rate between 0.1 and 10 °C / min (e.g., at least, at most, exactly, or between any two of 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1 °C / min, 1.1 °C / min, 1.2 °C / min, 1.3 °C / min, 1.4 °C / min, 1.5 °C / min, 1.6 °C / min, 1.7 °C / min, 1.8 °C / min, 1.9 °C / min, 2 °C / min, 2.1 °C / min, 2.2 °C / min, 2.3 °C / min, 2.4 °C / min, 2.5 °C / min, 2.6 °C / min, 2.7 °C / min, 2.8 °C / min, 2.9 °C / min, 3 °C / min, 3.1 °C / min, 3.2 °C / min, 3.3 °C / min, 3.4 °C / min, 3.5 °C / min, 3.6 °C / min, 3.7 °C / min, 3.8 °C / min, 3.9 °C / min, 4 °C / min,4.1 °C / min, 4.2 °C / min, 4.3 °C / min, 4.4 °C / min, 4.5 °C / min, 4.6 °C / min, 4.7 °C / min, 4.8 °C / min, 4.9 °C / min, 5 °C / min, 5.1 °C / min, 5.2 °C / min, 5.3 °C / min, 5.4 °C / min, 5.5 °C / min,5.6 °C / min, 5.7 °C / min, 5.8 °C / min, 5.9 °C / min, 6 °C / min, 6.1 °C / min, 6.2 °C / min, 6.3 °C / min,6.4 °C / min, 6.5 °C / min, 6.6 °C / min, 6.7 °C / min, 6.8 °C / min, 6.9 °C / min, 7 °C / min, 7.1 °C / min,7.2 °C / min, 7.3 °C / min, 7.4 °C / min, 7.5 °C / min, 7.6 °C / min, 7.7 °C / min, 7.8 °C / min, 7.9 °C / min, 8 °C / min, 8.1 °C / min, 8.2 °C / min, 8.3 °C / min, 8.4 °C / min, 8.5 °C / min, 8.6 °C / min,8.7 °C / min, 8.8 °C / min, 8.9 °C / min, 9 °C / min, 9.1 °C / min, 9.2 °C / min, 9.3 °C / min, 9.4 °C / min,9.5 °C / min, 9.6 °C / min, 9.7 °C / min, 9.8 °C / min, 9.9 °C / min, or 10 °C / min). In some instances, the temperature can be increased at a rate between 0.1 °C / min and 5 °C / min. Any one or more of the foregoing first drying step temperature rate increases may be expressly excluded from the methods described herein.

[0149] In some aspects, a temperature change associated with the first drying step of the tissue sample can be performed over a period of time, such as over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, or greater than 100 minutes. In some aspects, a temperature change associated with the first drying step of the tissue sample can be performed over about 20 minutes to about 60 minutes, about 30 minutes to about 50 minutes, or about 40 minutes. Any one or more of the foregoing time periods over which the first drying step is conducted may be expressly excluded from the methods described herein.

[0150] In some aspects, the first drying step of the tissue sample after freezing can be carried out for less than 10 hours. In some aspects, the first drying step of the tissue sample after freezing can be carried out for 10, 12, 14, 16, 18, 20, or 24 hours. In some aspects, the first drying step of the tissue sample after freezing can be carried out for 24, 48, or 72 hours. In some aspects, the first drying step of the tissue sample after freezing comprises holding the tissue at a noted temperature under a noted pressure for a specific period of time, ranging from 30 minutes to about 600 minutes. In some aspects, a first drying step comprises or consists of holding the tissue sample for at least, at most, about, exactly, or between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504,505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523,524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, or 600 minutes. In some aspects, a tissue sample can be held at a first drying temperature for about 300 minutes to about 420 minutes, about 320 minutes to about 400 minutes, about 340 minutes to about 380 minutes, or about 360 minutes. Any one or more of the foregoing times for which the tissue sample is held at the first drying step may be expressly excluded from the methods described herein. e. Performing A Second Drying Step Of The Tissue Sample After The First Drying Step

[0151] In some aspects, a second drying step can be carried out at a temperature that is greater than the temperature of the freezing step. In some aspects, the second drying step canbe carried out at a temperature that is greater than the temperature of the freezing step and the first drying step. In some aspects, prior to and / or starting with a second drying step, a vacuum pressure is initiated on the tissue sample. In some aspects, the second drying step of the tissue sample after the first drying step occurs at a vacuum SP (mTorr) of about 150 to 250, or about 175 to 225, or about 200 mTorr (e.g., an mTorr range of 150 to 250, e.g., at least, at most, exactly, or between any two of 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 mTorr). Any one or more of the foregoing second drying step vacuum SP values may be expressly excluded from the methods described herein.

[0152] In some aspects, the temperature is increased between the first drying step and the second drying step. In such aspects, the temperature of the second drying step is higher than the temperature of the first drying step. In some aspects, wherein the temperature of the second drying step is higher than the first drying step, the rate of the temperature increase from the first drying step can be gradual or rapid. For example, the rate of temperature increase from the first drying step to the second drying step can be from 0.1 to 5 °C / min (e.g., at least, at most, exactly, or between any two of 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1 °C / min, 1.1 °C / min, 1.2 °C / min, 1.3 °C / min,1.4 °C / min, 1.5 °C / min, 1.6 °C / min, 1.7 °C / min, 1.8 °C / min, 1.9 °C / min, 2 °C / min, 2.1 °C / min, 2.2 °C / min, 2.3 °C / min, 2.4 °C / min, 2.5 °C / min, 2.6 °C / min, 2.7 °C / min, 2.8 °C / min, 2.9 °C / min, 3 °C / min, 3.1 °C / min, 3.2 °C / min, 3.3 °C / min, 3.4 °C / min, 3.5 °C / min, 3.6 °C / min, 3.7 °C / min, 3.8 °C / min, 3.9 °C / min, 4 °C / min, 4.1 °C / min, 4.2 °C / min, 4.3 °C / min, 4.4 °C / min,4.5 °C / min, 4.6 °C / min, 4.7 °C / min, 4.8 °C / min, 4.9 °C / min, or 5 °C / min). In some aspects, the rate of temperature increase from the first drying step to the second drying step can be about 0.33 to 2 °C / min, or about 0.5 to 1.5 °C / min, or about 1 °C / min. Any one or more of the foregoing second drying step temperature rate increases may be expressly excluded from the methods described herein.

[0153] In some aspects, a temperature change associated with the second drying step of the tissue sample can be performed over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, or greater than 100 minutes. In some aspects, a temperature change associated with the second drying step of the tissue sample can be performed over about 5 minutes to about 25 minutes, about 10 minutes to about 20 minutes, or about 15 minutes. Any one or more of the foregoing time periods over which the second drying step is conducted may be expressly excluded from the methods described herein.

[0154] In some aspects, the second drying step can occur at a temperature of no more than 39 °C. In some aspects, the second drying step can occur at a temperature of no more than 45 Of

[0155] In some aspects, the second drying step of the tissue sample after the first drying step occurs between 5 °C and 25 °C. In some aspects, the second drying step of the tissue sample after the first drying step occurs between 10 °C and 20 °C. In some aspects, the second drying step of the tissue sample after the first drying step occurs between 12.5 °C and 17.5 °C.In some aspects, the second drying step of the tissue sample after the first drying step occurs at about 15 °C. In some aspects, the second drying step of the tissue sample after the first drying step raises the temperature from the first drying temperature to at least, at most, exactly, or between any two of -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C, -38 °C, -37 °C, -36°C, -35 °C, -34 °C, -33 °C, -32 °C, -31 °C, -30 °C, -29 °C, -28 °C, -27 °C, -26 °C, -25 °C, -24°C, -23 °C, -22 °C, -21 °C, -20 °C, -19 °C, -18 °C, -17 °C, -16 °C, -15 °C, -14 °C, -13 °C, -12°C, -11 °C, -10 °C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2°C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C,17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, or greater than 25 °C. In some aspects, the second drying step of the tissue sample after the first drying step comprises raising the temperature from the first drying temperature to between about 10 °C to about 20 °C, between about 12.5 °C to about 17.5 °C, or about 15 °C. Any one or more of the foregoing second drying step temperatures may be expressly excluded from the methods described herein.

[0156] In some aspects, the second drying step can be carried out at two or more different temperatures. In some aspects, the at least two different temperatures can be at least, at most, exactly, or between any two of 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, or 50 °C different from each other. For example, the second drying step can be carried out at 0 °C and then at 20 °C. In some aspects, the second drying step can be conducted in at least two different temperatures, wherein each different temperature can each be maintained for 5 to 15 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 6 min, 7 min, 8 min, 9min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min) each. In some aspects, the temperature can be ramped up from one temperature to the next, each of the intervening temperatures can be maintained for about 10 sec to 1 minute (e.g., at least, at most, exactly, or between any two of 10 sec, 11 sec, 12 sec, 13 sec, 14 sec, 15 sec, 16 sec, 17 sec, 18 sec, 19 sec, 20 sec, 21 sec,22 sec, 23 sec, 24 sec, 25 sec, 26 sec, 27 sec, 28 sec, 29 sec, 30 sec, 31 sec, 32 sec, 33 sec, 34 sec, 35 sec, 36 sec, 37 sec, 38 sec, 39 sec, 40 sec, 41 sec, 42 sec, 43 sec, 44 sec, 45 sec, 46 sec,47 sec, 48 sec, 49 sec, 50 sec, 51 sec, 52 sec, 53 sec, 54 sec, 55 sec, 56 sec, 57 sec, 58 sec, 59 sec, or 60 sec). Thus, although the second drying step can be carried out at two or more different temperatures, many temperatures can be involved in the second drying step as the tissue sample is exposed to all of the temperatures in between the at least two temperatures that are maintained for 5-15 minutes. Any one or more of the foregoing times and temperatures of the second drying step may be expressly excluded from the methods described herein.

[0157] In some aspects, the second drying step can be conducted at more than two different temperatures. For example, the second drying step can be conducted at 0 °C, 20 °C, and 30 °C. In some aspects, the second drying step can be conducted in at least three different temperatures, wherein each different temperature can be each maintained for 5 to 15 minutes each. As the temperature is ramped up from one temperature to the next, each of the intervening temperatures can be maintained for about 10 sec to 1 minute. Thus, although the second drying step can be carried out at three or more different temperatures, many temperatures can be involved in the second drying step as the tissue sample is exposed to all of the temperatures in between the at least two temperatures that are maintained for 5-15 minutes.

[0158] In some aspects, the second drying step is conducted for 12-144 hours. In some aspects, the second drying step is conducted for 12-48 hours. In some aspects, the second drying step is conducted for 12-72 hours. In some aspects, the second drying step is conducted for at least, at most, exactly, or between any two of 12, 15, 12, 25, 0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 144 hours. Any one or more of the foregoing times for which the second drying step is conducted may be expressly excluded from the methods described herein.

[0159] In some aspects, the second drying step of the tissue sample after the first drying step comprises holding the tissue at a noted temperature under a noted pressure for a specific period of time, ranging from 30 minutes to about 600 minutes, or greater than 600 minutes. In some aspects, a second drying step comprises or consists of holding the tissue sample for at least, at most, about, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508,509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565,566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, or 600 minutes. In some aspects, a tissue sample can be held at a second drying temperature for about 460 minutes to about 580 minutes, about 480 minutes to about 560 minutes, about 500 minutes to about 540 minutes, or about 520 minutes. Any one or more of the foregoing times for which the tissue sample is held at the second drying step may be expressly excluded from the methods described herein.

[0160] In some aspects, after drying the lyophilized tissue, the tissue can be cut to a desired size and / or shape. In some aspects, prior to drying the lyophilized tissue, the tissue is cut to a desired size and / or shape. f. Performing A Third Drying Step Of The Tissue Sample After The Second Drying Step

[0161] In some aspects, a third drying step can be carried out at a temperature that is greater than the temperature of the freezing step. In some aspects, the second drying step can be carried out at a temperature that is greater than the temperature of the freezing step, the first drying step, and the second drying step. In some aspects, prior to and / or starting with a third drying step, a vacuum pressure is initiated on the tissue sample. In some aspects, the third drying step of the tissue sample after the second drying step occurs at a vacuum SP (mTorr) of about 150 to 250, or about 175 to 225, or about 200 mTorr (e.g., an mTorr range of 150 to 250, e.g., at least, at most, exactly, or between any two of 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 mTorr). Any one or more of the foregoing vacuum SP values may be expressly excluded from the methods described herein.

[0162] In some aspects, the temperature is increased between the second drying step and the third drying step. In such aspects, the temperature of the third drying step is higher than the temperature of the second drying step. In some aspects, wherein the temperature of the third drying step is higher than the second drying step, the rate of the temperature increase from the second drying step can be gradual or rapid. For example, the rate of temperature increase from the second drying step to the third drying step can be from 0.1 to 5 °C / min (e.g., at least, at most, exactly, or between any two of 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1 °C / min, 1.1 °C / min, 1.2 °C / min,1.3 °C / min, 1.4 °C / min, 1.5 °C / min, 1.6 °C / min, 1.7 °C / min, 1.8 °C / min, 1.9 °C / min, 2 °C / min, 2.1 °C / min, 2.2 °C / min, 2.3 °C / min, 2.4 °C / min, 2.5 °C / min, 2.6 °C / min, 2.7 °C / min, 2.8 °C / min, 2.9 °C / min, 3 °C / min, 3.1 °C / min, 3.2 °C / min, 3.3 °C / min, 3.4 °C / min, 3.5 °C / min, 3.6 °C / min, 3.7 °C / min, 3.8 °C / min, 3.9 °C / min, 4 °C / min, 4.1 °C / min, 4.2 °C / min, 4.3 °C / min,4.4 °C / min, 4.5 °C / min, 4.6 °C / min, 4.7 °C / min, 4.8 °C / min, 4.9 °C / min, or 5 °C / min). In some aspects, the rate of temperature increase from the second drying step to the third drying stepcan be about 0.33 to 2 °C / min, or about 0.5 to 1.5 °C / min, or about 1 °C / min. Any one or more of the foregoing third drying step temperature rate increases may be expressly excluded from the methods described herein.

[0163] In some aspects, a temperature change associated with the third drying step of the tissue sample can be performed over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100, or greater than 100 minutes. In some aspects, a temperature change associated with the third drying step of the tissue sample can be performed over about 2 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 10 minutes. Any one or more of the foregoing time periods over which the third drying step is conducted may be expressly excluded from the methods described herein.

[0164] In some aspects, the third drying step of the tissue sample after the second drying step occurs between 15 °C and 35 °C. In some aspects, the third drying step of the tissue sample after the second drying step occurs between 20 °C and 30 °C. In some aspects, the third drying step of the tissue sample after the second drying step occurs between 22.5 °C and 27.5 °C. In some aspects, the third drying step of the tissue sample after the second drying step occurs at about 25 °C. In some aspects, the third drying step of the tissue sample after the second drying step raises the temperature from the second drying temperature to at least, at most, exactly, or between any two of -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C, -38 °C, -37 °C, -36°C, -11 °C, -10 °C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2°C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C,17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30°C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, or greater than 35 °C. In some aspects, the third drying step of the tissue sample after the second drying step comprises raising the temperature from the second drying temperature to between about 20 °C to about 30 °C, between about 22.5 °C to about 27.5 °C, or about 25 °C. Any one or more of the foregoing third drying step temperatures may be expressly excluded from the methods described herein.

[0165] In some aspects, the third drying step is conducted for 12-144 hours. In some aspects, the third drying step is conducted for 12-48 hours. In some aspects, the third dryingstep is conducted for 12-72 hours. In some aspects, the third drying step is conducted for at least at most, exactly, or between any two of 12, 15, 12, 25, 0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 144 hours. Any one or more of the foregoing time periods over which the third drying step is conducted may be expressly excluded from the methods described herein.

[0166] In some aspects, the third drying step of the tissue sample after the second drying step comprises holding the tissue at a noted temperature under a noted pressure for a specific period of time, ranging from 30 minutes to about 600 minutes, or greater than 600 minutes. In some aspects, a third drying step comprises or consists of holding the tissue sample for at least, at most, about, exactly, or between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550,551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, or 600 minutes. In some aspects, a tissue sample can be held at a third drying temperature for about 300 minutes to about 420 minutes, about 320 minutes to about 400 minutes, about 340 minutes to about 380 minutes, or about 360 minutes. Any one or more of the foregoing times for which the tissue sample is held at the third drying step may be expressly excluded from the methods described herein.

[0167] In some aspects, after drying the lyophilized tissue, the tissue can be cut to a desired size and / or shape. In some aspects, prior to drying the lyophilized tissue, the tissue is cut to a desired size and / or shape. g. Performing A Fourth Drying Step Of The Tissue Sample After The Third Drying Step

[0168] In some aspects, a fourth drying step can be carried out at a temperature that is greater than the temperature of the freezing step. In some aspects, the fourth drying step can be carried out at a temperature that is greater than the temperature of the freezing step, the first drying step, the second drying step, and optionally greater than the third drying step. In some aspects, prior to and / or starting with a fourth drying step, a vacuum pressure is initiated on the tissue sample. In some aspects, the fourth drying step of the tissue sample after the third drying step occurs at a vacuum SP (mTorr) of about 25 to about 200, about 50 to about 150, about 75 to about 125, or about 100 mTorr (e.g., an mTorr range of 25 to 200, e.g., at least, at most, exactly, or between any two of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, or 250 mTorr). In some aspects, the fourth drying step of the tissue sample after the third drying step occurs at a vacuum SP (mTorr) lower than one or more, or all, of the preceding drying steps. Any one or more of the foregoing vacuum SP values may be expressly excluded from the methods described herein.

[0169] In some aspects, the temperature is increased between the third drying step and the fourth drying step. In such aspects, the temperature of the fourth drying step is higher than the temperature of the third drying step. In some aspects, wherein the temperature of the fourth drying step is higher than the third drying step, the rate of the temperature increase from the third drying step can be gradual or rapid. For example, the rate of temperature increase from the third drying step to the fourth drying step can be from 0.1 to 5 °C / min (e.g., at least, at most, exactly, or between any two of 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1 °C / min, 1.1 °C / min, 1.2 °C / min, 1.3 °C / min,1.4 °C / min, 1.5 °C / min, 1.6 °C / min, 1.7 °C / min, 1.8 °C / min, 1.9 °C / min, 2 °C / min, 2.1 °C / min, 2.2 °C / min, 2.3 °C / min, 2.4 °C / min, 2.5 °C / min, 2.6 °C / min, 2.7 °C / min, 2.8 °C / min, 2.9 °C / min, 3 °C / min, 3.1 °C / min, 3.2 °C / min, 3.3 °C / min, 3.4 °C / min, 3.5 °C / min, 3.6 °C / min, 3.7 °C / min, 3.8 °C / min, 3.9 °C / min, 4 °C / min, 4.1 °C / min, 4.2 °C / min, 4.3 °C / min, 4.4 °C / min,4.5 °C / min, 4.6 °C / min, 4.7 °C / min, 4.8 °C / min, 4.9 °C / min, or 5 °C / min). In some aspects, the rate of temperature increase from the third drying step to the fourth drying step can be about 0.33 to 2 °C / min, or about 0.5 to 1.5 °C / min, or about 1 °C / min. In some aspects, there is no temperature increase between the third drying step and the fourth drying step. Any one or more of the foregoing fourth drying step temperature rate increases may be expressly excluded from the methods described herein.

[0170] In some aspects, a temperature change associated with the fourth drying step of the tissue sample can be performed over a period of time, such as over at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100, or greater than 100 minutes. In some aspects, a temperature change associated with the fourth drying step of the tissue sample can be performed over about 2 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 10 minutes. Any one or more of the foregoing time periods over which the fourth drying step is conducted may be expressly excluded from the methods described herein.

[0171] In some aspects, the fourth drying step of the tissue sample after the third drying step occurs between 15 °C and 35 °C. In some aspects, the fourth drying step of the tissue sample after the third drying step occurs between 20 °C and 30 °C. In some aspects, the fourth drying step of the tissue sample after the third drying step occurs between 22.5 °C and 27.5 °C. In some aspects, the fourth drying step of the tissue sample after the third drying step occurs at about 25 °C. In some aspects, the fourth drying step of the tissue sample after the third drying step raises the temperature from the third drying temperature to at least, at most, exactly, or between any two of -45 °C, -44 °C, -43 °C, -42 °C, -41 °C, -40 °C, -39 °C, -38 °C, -37 °C, -36°C, -35 °C, -34 °C, -33 °C, -32 °C, -31 °C, -30 °C, -29 °C, -28 °C, -27 °C, -26 °C, -25 °C, -24°C, -23 °C, -22 °C, -21 °C, -20 °C, -19 °C, -18 °C, -17 °C, -16 °C, -15 °C, -14 °C, -13 °C, -12°C, -11 °C, -10 °C, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, 0 °C, 1 °C, 2°C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C,17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30°C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, or greater than 35 °C. In some aspects, the fourth drying step of the tissue sample after the third drying step comprises raising the temperature from the third drying temperature to between about 20 °C to about 30 °C, between about 22.5 °C to about 27.5 °C, or about 25 °C. in some aspects, the fourth drying step of the tissue sample after the third drying step does not comprise raising the temperature from the third drying step. Any one or more of the foregoing fourth drying step temperatures may be expressly excluded from the methods described herein.

[0172] In some aspects, the fourth drying step is conducted for 12-144 hours. In some aspects, the fourth drying step is conducted for 12-48 hours. In some aspects, the fourth drying step is conducted for 12-72 hours. In some aspects, the fourth drying step is conducted for at least, at most, exactly, or between any two of 12, 15, 12, 25, 0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 144 hours. Any one or more of the foregoing time periods over which the fourth drying step is conducted may be expressly excluded from the methods described herein.

[0173] In some aspects, the fourth drying step of the tissue sample after the third drying step comprises holding the tissue at a noted temperature under a noted pressure for a specific period of time, ranging from 0 minutes to about 100 minutes, or greater than 100 minutes. In some aspects, a fourth drying step comprises or consists of holding the tissue sample for at least, at most, about, exactly, or between 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or greater than 100 minutes. In some aspects, a tissue sample can be held at a fourth drying temperature for about 1 minutes to about 40 minutes, about 5 minutes to about 35 minutes, about 10 minutes to about 30 minutes, or about 20 minutes.

[0174] In some aspects, a tissue sample can be stored after the fourth drying step at the fourth drying temperature until vacuum is released. In some aspects, a tissue sample can be stored after the fourth drying step at the fourth drying temperature for any period of time, for example but not limited to, 1, 2, 3, 4, 5, 6, 7, or greater than 7, hours, days, weeks, or months. Any one or more of the foregoing times for which the tissue sample is held at the fourth drying step may be expressly excluded from the methods described herein.

[0175] In some aspects, after drying the lyophilized tissue, the tissue can be cut to a desired size and / or shape. In some aspects, prior to drying the lyophilized tissue, the tissue is cut to a desired size and / or shape. h. Exemplary Lyophilization Process

[0176] For preparation of lyophilized placental tissue dressing, placental tissue samples can be received and rinsed, followed by a first quality control process. Blood vessels may be removed from the placental tissues. The placental tissue may undergo chemical treatment. After chemical treatment processing is completed, the tissue may be washed with PBS (e.g., IX, 2X, 3X, 4X, or 5X concentrated) and / or water. Each washing step may take about 1 minute to 60 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min). For sheet or foam forms of dressing, placental tissue may be washed with three repeated changes of concentrated PBS (e.g., IX, 2X, 3X, 4X, or 5X concentrated) and / or water, with each change including rinsing with concentrated (e.g., IX, 2X, 3X, 4X, or 5X concentrated) PBS for about 1 minute to 60 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min) followed by rinsing with water for about 1 minute to 60 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min). For a particulate form of dressing, placental tissue may be washed with five repeated changes of concentrated PBS and / or water with each change including rinsing with concentrated PBS for about 1 minute to 60 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min)followed by rinsing with water for about 1 minute to 60 minutes (e.g., at least, at most, exactly, or between any two of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min,35 min, 40 min, 45 min, 50 min, 55 min, or 60 min). The washing process allows for removal chemical treatment residue. Subsequently, the placental tissue may be subject to additional washing steps, including washing with water for 12 to 36 hours (e.g., at least, at most, exactly, or between any two of 12 hrs, 14 hrs, 16 hrs, 18 hrs, 20 hrs, 22 hrs, 24 hrs, 26 hrs, 28 hrs, 30 hrs, 32 hrs, 34 hrs, or 36 hrs) and another washing step with water for 1 hour to 3 hours (e.g., at least, at most, exactly, or between any two of 1, 1.1 hrs, 1.2 hrs, 1.3 hrs, 1.4 hrs, 1.5 hrs, 1.6 hrs, 1.7 hrs, 1.8 hrs, 1.9 hrs, 2 hrs, 2.1 hrs, 2.2 hrs, 2.3 hrs, 2.4 hrs, 2.5 hrs, 2.6 hrs, 2.7 hrs, 2.8 hrs, 2.9 hrs, or 3 hrs) followed by an endotoxin test. After completion of the washing steps, the tissue may undergo freezing at -70°C for overnight and be subject to a first packaging process. The tissue may then go through a lyophilization process, followed by a second quality control process. Next, the lyophilized tissue may be cut into appropriate sizes, followed by a second and a third packaging process. The lyophilized umbilical tissue may be sterilized (e.g., by E- beam), including causing viral inactivation, followed by a third quality control process. The placental tissue may be cut into appropriate sizes prior to or after lyophilization.

[0177] In an exemplary method, preparation of placental tissue and / or lyophilization of portions of said tissue (e.g., amnion, chorion, and / or trophoblast) can comprise: 1. Thawing the frozen donor tissues (e.g., whole placenta including umbilical cord (UC)) at ambient temperature. 2. Cutting the UC from the placenta and transfer the UC to the separate container. 3. Removing the amnion from chorion and decidua by hand and transferring the amnion to a separate container comprising PBS for products utilizing amnion. 4. Removing the chorion from decidua by hand and transferring the chorion to a separate container comprising PBS for products comprising chorionic mesoderm only and / or a chorionic mesoderm and trophoblast layer; in aspects comprising chorionic mesoderm only, the trophoblast layer can be removed, for example via dispase incubation. 5. Rinsing the placental tissue with PBS to remove blood and jelly. 6. Placing each placental tissue sample into an antibiotics solution and incubating of the samples at about 37 °C for 12 hours to 24 hours (e.g., at least, at most, exactly, or between any two of 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, or 24 hrs) without shaking. 7. Collecting the incubated placental tissue samples from the antibiotics solution and rinsing them with PBS. 8. Cutting the placental tissue with a blade to make pieces of the appropriate size (e.g., 3 cm x 4 cm, 3 cm x 8 cm, etc.) 9. Attaching the cut placental tissue to a backing. 10. Covering the placental tissue with another backing if necessary. 11. Drying excess moisture from the sample prior to packaging. 12. Placing samples with backing meshes into Tyvek-backed pouches and sealing the pouches. 13. Placing the pouches into a foil pouch with Tyvek headers and sealing along the edges of the Tyvek header.14. Placing the sealed foil pouches within a suitable lyophilizer unit. 15. Running the lyophilizer unit with the protocol provided in Table 1, with a final storage / hold of 25 °C with Vac SP (mTorr) 100.Table 1 - Exemplary lyophilization cycle

[0178] In some aspects, at no point during the aforementioned steps are the samples contacted with a lyoprotectant and / or a lyoprotectant comprising trehalose.V. PLACENTAL TISSUE COMPOSITIONS

[0179] In some aspects, a placental tissue compositions can include cryopreserved or dehydrated whole placenta or portion of a placenta. In some aspects, a placental tissue compositions can include cryopreserved or dehydrated chorion, amnion, a chorion and amniotic membrane (e.g., amnio-chorion), Wharton’s jelly, umbilical cord, placental cotyledons or combinations thereof. In some aspects, a placental tissue compositions can include cryopreserved or dehydrated placental tissue that has been dissected or digested (or combinations thereof) to remove portions, membrane, or structures. In some aspects, placental tissue compositions are used in a purposeful wound dressing product with superior biological properties with respect to supporting the wound healing process by allowing cellular proliferation and migration, vascular ingrowth, and / or the formation of granulation tissues.

[0180] In some aspects, the cryopreserved or dehydrated tissue disclosed herein can be stable for at least three weeks. In some aspects, the cryopreserved or dehydrated tissue can be stable for at least three months. In some aspects, the cryopreserved or dehydrated tissue can be stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 60 months.

[0181] In some aspects, the cryopreserved or dehydrated tissue disclosed herein can be thawed or reconstituted. Cryopreserved tissue can be thawed using standard techniques known in the art. Dehydrated tissue can be reconstituted using standard techniques known in the art. In some aspects, reconstituting refers to rehydrating. Thus, the disclosed dehydrated tissues canbe reconstituted or rehydrated using water, saline, a buffer such as, but not limited to phosphate buffered saline (PBS), in a solution comprising a stabilizing agent such as, but not limited to bovine serum albumin (BSA), Plasma-Lyte A or other clinically available electrolyte solutions, with human bodily fluids or a combination thereof. For example, dehydrated tissue can be applied directly to a wound or tissue injury on a subject and the subject's bodily fluids can reconstitute. In some aspects, a combination of bodily fluids and another known rehydrating solution can be used.

[0182] The thawed or reconstituted tissue derived from the methods disclosed herein can comprise native viable cells and native therapeutic factors. The thawed or reconstituted tissue can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% viable cells compared to the same tissue prior to cryopreservation or dehydration. The thawed or reconstituted tissue can comprise at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% viable native cells compared to the same tissue prior to cryopreservation or dehydration. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein comprise native viable cells and native therapeutic factors. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein do not comprise native viable cells but comprise native therapeutic factors. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein are not decellularized. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein are not decellularized but do not comprise native viable cells. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein are decellularized. In some aspects, the thawed or reconstituted tissue derived from the methods disclosed herein are decellularized but comprise native therapeutic factors.

[0183] In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue, e.g., air-dried or lyophilized placental tissue, or compositions thereof, may be in the form of a laminate or multi-layer placental tissue. As shown in FIG. 2, the laminate may comprise one or more placental tissues having a first layer opposite a second layer, and the one or more placental tissues may be in the form of sheets. FIG. 2 depicts a single layer of placental tissue 10a used to produce a layered placental tissue laminate 10b. The single layer placental tissue 10a includes a first layer I la and a second layer 11b. The placental tissue laminate 10b includes two layers or sheets of placental tissue 10a, each layer or sheet of placental tissue 10a having a single first layer I la and a single second layer 11b.

[0184] The laminate may be formed by folding one of the one or more placental tissues to contact the first layer of a first portion of the placental tissue with the first layer of a secondportion of the placental tissue. Additionally, or alternatively, the laminate may be formed by layering a first of the placental tissues with a second of the placental tissues by contacting the first layer of the first of the placental tissues with the first layer of the second of the placental tissues. In this way, a placental tissue graft (e.g., an amniotic tissue graft) is produced having layers of a first type (e.g., an epithelial layer) external to each side the graft and layers of a second type (e.g., a stromal layer) internal to the graft.

[0185] The laminate may comprise 1, 2, 3, 4, 5, or more placental tissues and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more placental tissue layers. Each placental tissue of the laminate may have a thickness of 0.1 to 100 pM, e.g., at least, at most, exactly, or between any two of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pM. The laminate may have a total thickness of 1 to 1000 pM, e.g., at least, at most, exactly, or between any two of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, or 1000 pM.

[0186] In some aspects, the laminate may comprise a middle or interior layer, e.g., an additional layer comprised or sandwiched between the placental tissues or placental tissue layers and contacting at least a portion of the placental tissues or placental tissue layers. The middle layer may be a reinforcing material, e.g., to support the structural or mechanical integrity of the placental tissue graft. The middle layer may comprise any suitable reinforcing material, e.g., xenografts, allografts, other ECM-based tissues, porcine small intestinal submucosa (SIS), collagen or synthetic / bio- synthetic materials (e.g., PLA / PGA / polydioxanone mesh or mat), etc.

[0187] In some aspects, a placental tissue graft is manufactured by obtaining one or more placental tissues, each placental tissue having a first layer opposite a second layer, and forming a placental tissue laminate by (1) folding one of the one or more placental tissues to contact the first layer of a first portion of the placental tissue with the first layer of a second portion of the placental tissue; or (2) layering a first of the placental tissues with a second of the placental tissues by contacting the first layer of the first of the placental tissues with the first layer of the second of the placental tissues.

[0188] In some aspects, a placental tissue graft is manufactured by obtaining one or more placental tissues, each placental tissue having a first layer opposite a second layer, and forminga placental tissue laminate by (1) folding one of the one or more placental tissues to contact the second layer of a first portion of the placental tissue with the second layer of a second portion of the placental tissue; or (2) layering a first of the placental tissues with a second of the placental tissues by contacting the second layer of the first of the placental tissues with the second layer of the second of the placental tissues.

[0189] In some aspects, a placental tissue graft is manufactured by obtaining one or more placental tissues, each placental tissue having a first layer opposite a second layer, and forming a placental tissue laminate by (1) folding one of the one or more placental tissues to contact the first layer of a first portion of the placental tissue with the second layer of a second portion of the placental tissue; or (2) layering a first of the placental tissues with a second of the placental tissues by contacting the first layer of the first of the placental tissues with the second layer of the second of the placental tissues.

[0190] In some aspects, an amniotic tissue graft is manufactured by obtaining one or more amniotic tissues, each amniotic tissue having a stromal layer opposite an epithelial layer, and forming a amniotic tissue laminate by (1) folding one of the one or more amniotic tissues to contact the stromal layer of a first portion of the amniotic tissue with the stromal layer of a second portion of the amniotic tissue; or (2) layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the stromal layer of the first of the amniotic tissues with the stromal layer of the second of the amniotic tissues.

[0191] In some aspects, an amniotic tissue graft is manufactured by obtaining one or more amniotic tissues, each amniotic tissue having a stromal layer opposite an epithelial layer, and forming a amniotic tissue laminate by (1) folding one of the one or more amniotic tissues to contact the epithelial layer of a first portion of the amniotic tissue with the epithelial layer of a second portion of the amniotic tissue; or (2) layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the epithelial layer of the first of the amniotic tissues with the epithelial layer of the second of the amniotic tissues.

[0192] In some aspects, an amniotic tissue graft is manufactured by obtaining one or more amniotic tissues, each amniotic tissue having a stromal layer opposite an epithelial layer, and forming a amniotic tissue laminate by (1) folding one of the one or more amniotic tissues to contact the stromal layer of a first portion of the amniotic tissue with the epithelial layer of a second portion of the amniotic tissue; or (2) layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the stromal layer of the first of the amniotic tissues with the epithelial layer of the second of the amniotic tissues.

[0193] In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue, e.g., air-dried or lyophilized placental tissue, may be in the form of a sheet, pieces, or particulate. In some aspects, the placental tissue is cryopreserved, optionally, decellularized and / or non- viable, and in particulate, sheet, and / or foam form. In some aspects, the placental tissue is dehydrated, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form. In some aspects, placental tissue comprises, consists, or consists essentially of any mixture composed of birth tissues.

[0194] In some aspects, the placental tissue comprises amniotic membrane or amniotic membrane tissue. In some aspects, the placental tissue comprises, consists, or consists essentially of amnion. In some aspects, the placental tissue comprises, consists, or consists essentially of double layered amnion or amniotic membrane. In some aspects, the amniotic membrane or amniotic tissue is cryopreserved, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form. In some aspects, the amniotic membrane or amniotic tissue is dehydrated, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form.

[0195] In some aspects, the placental tissue comprises chorionic membrane or chorionic membrane tissue. In some aspects, the placental tissue comprises, consists, or consists essentially of chorion. In some aspects, the placental tissue comprises, consists, or consists essentially of chorion with trophoblast. In some aspects, the placental tissue comprises, consists, or consists essentially of double layered chorion or chorionic membrane. In some aspects, the chorionic membrane or chorionic tissue is cryopreserved, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form. In some aspects, the chorionic membrane or chorionic tissue is dehydrated, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form.

[0196] In some aspects, the placental tissue comprises, consists, or consists essentially of amnion with chorion. In some aspects, the amnion and chorion are layered. In some aspects, the amnion and chorion are cryopreserved, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form. In some aspects, the amnion and chorion are dehydrated, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form.

[0197] In some aspects, placental tissue comprises, consists, or consists essentially of umbilical cord. In some aspects, the umbilical cord is layered. In some aspects, the umbilical cord is cryopreserved, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form. In some aspects, the umbilical cord is dehydrated, optionally, decellularized and / or non-viable, and in particulate, sheet, and / or foam form.

[0198] In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam is non-immunogenic and / or immunoprivileged. In some aspects, the compositions or cryopreserved or dehydrated compositions disclosed herein comprising placental tissue or cryopreserved or dehydrated placental tissue may be non- immunogenic and / or immunoprivileged. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam comprises native viable cells. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam does not comprise viable cells. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam is not decellularized. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam is not decellularized but does not comprise viable cells. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet, and / or foam is decellularized.

[0199] In some aspects, the cryopreserved or dehydrated, e.g., air-dried or lyophilized, placental tissue particulate may have a particle size of 90% less than 250 microns. The cryopreserved or dehydrated placental tissue particulate may have a particle size or particle size distribution (mean particle size) of from about 425 to about 1000 microns, or from about 216 to about 425 microns, or less than about 216 microns. The cryopreserved or dehydrated placental tissue particulate may have a particle size of from about 1 to about 1000 microns, or from about 1 to about 500 microns, or from about 1 to about 250 microns, or from about 50 to about 1000 microns, or from about 50 to about 500 microns, or from about 50 to about 250 microns, or from about 100 to about 1000 microns, or from about 100 to about 500 microns, or from about 100 to about 250 microns, or from about 200 to about 1000 microns, or from about 200 to about 500 microns, or from about 200 to about 250 microns, or from about 400 to about 1000 microns, or from about 400 to about 500 microns. The particle size may be determined by particle sizing methods known to one of skill in the art such as, but not limited to microscopic analysis, stacked sieving methods, and particle size analyzers. Particle size analyzers may employ light obscuration methods or laser diffraction methods. Suitable particle size analyzers include but are not limited to analyzers made by Malvern such as the Malvern Mastersizer™, AccuSizer, Shimadzu Corporation, and Beckman Coulter. A particle size distribution may be expressed as a mean particle size based on number distribution or volume distribution.

[0200] In some aspects, the concentration of placental tissue, cryopreserved placental tissue, dehydrated placental tissue, lyophilized placental tissue, and / or dried placental tissue in the composition may be at an amount effective to provide an environment to support the wound healing process by allowing cellular migration, vascular ingrowth, and the formation of granulation tissues when the composition is applied to a wound or damaged tissue as demonstrated by the examples disclosed herein.

[0201] In some aspects, the concentrations of placental tissue, cryopreserved placental tissue, dehydrated placental tissue, lyophilized placental tissue, and / or dried placental tissue in the compositions in the hydrous state disclosed herein may be from about 0.05% to about 50% w / w, or from about 0.05% to about 40% w / w, or from about 0.05% to about 30% w / w, or from about 0.05% to about 25% w / w, or from about 0.05% to about 20% w / w, or from about 0.05% to about 15% w / w, or from about 0.05% to about 10% w / w, or from about 0.05% to about 5% w / w, or from about 0.05% to about 4% w / w, or from about 0.05% to about 3% w / w, or from about 0.05% to about 2% w / w, or from about 0.05% to about 1% w / w, or from about 0.1% to about 50% w / w, or from about 0.1% to about 40% w / w, or from about 0.1% to about 30% w / w, or from about 0.1% to about 25% w / w, or from about 0.1% to about 20% w / w, or from about 0.1% to about 15% w / w, or from about 0.1% to about 10% w / w, or from about 0.1% to about 5% w / w, or from about 0.1% to about 4% w / w, or from about 0.1% to about 3% w / w, or from about 0.1% to about 2% w / w, or from about 0.1% to about 1% w / w, or from about 0.5% to about 50% w / w, or from about 0.5% to about 40% w / w, or from about 0.5% to about 30% w / w, or from about 0.5% to about 25% w / w, or from about 0.5% to about 20% w / w, or from about 0.5% to about 15% w / w, or from about 0.5% to about 10% w / w, or from about 0.5% to about 5% w / w, or from about 0.5% to about 4% w / w, or from about 0.5% to about 3% w / w, or from about 0.5% to about 2% w / w, or from about 0.5% to about 1% w / w, or from about 1% to about 50% w / w, or from about 1% to about 40% w / w, or from about 1% to about 30% w / w, or from about 1% to about 25% w / w, or from about 1% to about 20% w / w, or from about 1% to about 15% w / w, or from about 1% to about 10% w / w, or from about 1% to about 5% w / w, or from about 1% to about 4% w / w, or from about 1% to about 3% w / w, or from about 1% to about 2% w / w, of the total composition for compositions in the hydrous state.

[0202] In some aspects, the concentrations of placental tissue, cryopreserved placental tissue, dehydrated placental tissue, lyophilized placental tissue, and / or dried placental tissue in the compositions in the cryopreserved or dehydrated state disclosed herein may be from about 1% to about 75% w / w, or from about 1% to about 60% w / w, or from about 1% to about 50% w / w, or from about 2% to about 50% w / w, or from about 3% to about 50% w / w, or from about4% to about 50% w / w, or from about 5% to about 50% w / w, or from about 6% to about 50% w / w, or from about 7% to about 50% w / w, or from about 8% to about 50% w / w, or from about 9% to about 50% w / w, or from about 10% to about 50% w / w, or from about 15% to about 50% w / w, or from about 20% to about 50% w / w, or from about 1% to about 40% w / w, or from about 2% to about 40% w / w, or from about 3% to about 40% w / w, or from about 4% to about 40% w / w, or from about 5% to about 40% w / w, or from about 6% to about 40% w / w, or from about 7% to about 40% w / w, or from about 8% to about 40% w / w, or from about 9% to about 40% w / w, or from about 10% to about 40% w / w, or from about 15% to about 40% w / w, or from about 20% to about 40% w / w, or from about 1% to about 30% w / w, or from about 2% to about 30% w / w, or from about 3% to about 30% w / w, or from about 4% to about 30% w / w, or from about 5% to about 30% w / w, or from about 6% to about 30% w / w, or from about 7% to about 30% w / w, or from about 8% to about 30% w / w, or from about 9% to about 30% w / w, or from about 10% to about 30% w / w, or from about 15% to about 30% w / w, or from about 20% to about 30% w / w, of the total composition for compositions in the dehydrated state.

[0203] In some aspects, placental tissue may be processed as described in the following non-limiting exemplified aspect. After being processed by the methods disclosed herein, the processed placental tissue may be cryopreserved or lyophilized (freeze-dried) using a lyophilizer or other suitable freeze-drying technique. In some aspects, the lyophilized placental tissue may be processed into a particulate form using a suitable mill, grinder, or dissociator. In some aspects, the lyophilized placental tissue may be processed into a sheet and / or foam form. In some aspects, the placental tissue, cryopreserved placental tissue, or dehydrated placental tissue particulate, sheet and / or foam may be decellularized and / or disinfected (e.g., viral inactivation) according to the methods disclosed herein.

[0204] In another aspect, disclosed are methods of producing compositions comprising placental tissue, wherein the method comprises manipulating the placental tissue. Manipulating placental tissue may comprise cutting, rolling, shaping, smoothing, or flattening the placental tissue. In some instances, manipulating placental tissue may comprise flattening the placental tissue. The rolling or flattening of the placental tissue may be performed with a laminating roller, such as a bubble buster, or a microdermal roller. Manipulating the placental tissue may result in the elevated release of native growth factors. Flattening of the umbilical tissue may provide a uniform thickness of the final product.

[0205] In some aspects, disclosed are methods of producing compositions comprising placental tissue, optionally comprising forming engineered channels in the placental tissue, further optionally comprising treating the placental tissue with at least one antibiotic. In someinstances, treating with at least one antibiotic comprises incubating the placental tissue with an antibiotic cocktail solution for 18 to 96 hours. In some instances, the placental tissue may be treated with two or more antibiotics simultaneously or consecutively. In some instances, the treatment with at least one antibiotic can be performed prior to cryopreservation. In some instances, the placental tissue may be cryopreserved in a solution comprising at least one antibiotic.

[0206] In some aspects, disclosed are methods of producing compositions comprising placental tissue, optionally comprising forming engineered channels in the placental tissue, and further optionally comprising cutting the placental tissue to a desired size. In some instances, cutting the placental tissue to a desired size comprises placing a cutter onto the placental tissue; and cutting the placental tissue to maintain a square shape. A cutter may be any device such as, but not limited to, a stencil that helps cut the tissue. A cutter may hold the tissue in place or provide a specific shape for the tissue to be cut to.

[0207] In some aspects, disclosed are methods of producing compositions comprising placental tissue, optionally comprising forming engineered channels in the placental tissue, and further optionally comprising inspecting the placental tissue for excess strings of tissue and discoloration.A. CARRIERS

[0208] The compositions comprising placental tissue disclosed herein may further comprise a carrier, such as a pharmaceutically acceptable carrier. Non-limiting examples of carriers include lotions, creams, emulsions, ointments, gels, hydrogels, pastes, solutions, aerosol sprays, aerosol foams, non-aerosol sprays, non-aerosol foams, powders, liquid solutions, liquid suspensions, films, and sheets. The carrier or pharmaceutically acceptable carrier may be aqueous based, anhydrous, hydrophilic, hydrophobic, or anhydrous hydrophilic. For example, an aqueous based carrier or pharmaceutically acceptable carrier may be an aqueous based gel or hydrogel. The compositions may be prepared by incorporating the placental tissue into the carrier using methods known to those of skill in the art. Such methods may include the use of various types of mixers, blenders, and homogenizers. The compositions of placental tissue and a carrier may be impregnated in gauzes, bandages, or other wound dressing materials. In some aspects, the placental tissue, dehydrated placental tissue, lyophilized placental tissue, or dried placental tissue is in the form of pieces or particulate and is distributed uniformly within the carrier. In some aspects, the carrier is a pharmaceutically acceptable carrier. In some aspects, the carrier or the pharmaceutically acceptable carrier isaqueous based. The compositions may be aqueous based, anhydrous, or dehydrated. Nonlimiting examples of dehydrated compositions comprising placental tissue and a carrier include films, sheets, foams, or powders. In some aspects of the present disclosure, an aqueous-based composition, carrier, gel, or hydrogel may include at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% w / w or more of water, preferably at least 50% w / w water.

[0209] In another aspect, compositions in the form of films or sheets are disclosed. The films or sheets may comprise placental in pieces or in particulate form, and a carrier. The terms “films” and “sheets” as used herein may be used interchangeably, but generally a film is thinner than a sheet. The thickness of the films or sheets is a function of the amount of the hydrogel placed into a mold prior to dehydration, i.e., the more hydrogel in a given mold, will result in a thicker film or sheet after dehydrating. The thickness of the dehydrated film or sheet may be from about 0.1 mm to about 25 mm, or from about 0.1 to about 20 mm, or from about 0.1 to about 15 mm, or from about 0.1 to about 10 mm, or from about 0.1 to about 9 mm, or from about 0.1 to about 8 mm, or from about 0.1 to about 7 mm, or from about 0.1 to about 6 mm, or from about 0.1 to about 5 mm, or from about 0.1 to about 4 mm, or from about 0.1 to about3 mm, or from about 0.1 to about 2 mm, or from about 0.1 to about 1.5 mm, or from about 0.1 to about 1 mm, or from about 0.2 mm to about 25 mm, or from about 0.2 to about 20 mm, or from about 0.2 to about 15 mm, or from about 0.2 to about 10 mm, or from about 0.2 to about9 mm, or from about 0.2 to about 8 mm, or from about 0.2 to about 7 mm, or from about 0.2 to about 6 mm, or from about 0.2 to about 5 mm, or from about 0.2 to about 4 mm, or from about 0.2 to about 3 mm, or from about 0.2 to about 2 mm, or from about 0.2 to about 1.5 mm, or from about 0.2 to about 1 mm, or from about 0.3 mm to about 25 mm, or from about 0.3 to about 20 mm, or from about 0.3 to about 15 mm, or from about 0.3 to about 10 mm, or from about 0.3 to about 9 mm, or from about 0.3 to about 8 mm, or from about 0.3 to about 7 mm, or from about 0.3 to about 6 mm, or from about 0.3 to about 5 mm, or from about 0.3 to about4 mm, or from about 0.3 to about 3 mm, or from about 0.3 to about 2 mm, or from about 0.3 to about 1.5 mm, or from about 0.3 to about 1 mm, or from about 0.4 mm to about 25 mm, or from about 0.4 to about 20 mm, or from about 0.4 to about 15 mm, or from about 0.4 to about10 mm, or from about 0.4 to about 9 mm, or from about 0.4 to about 8 mm, or from about 0.4 to about 7 mm, or from about 0.4 to about 6 mm, or from about 0.4 to about 5 mm, or from about 0.4 to about 4 mm, or from about 0.4 to about 3 mm, or from about 0.4 to about 2 mm, or from about 0.4 to about 1.5 mm, or from about 0.4 to about 1 mm, or from about 0.5 mm to about 25 mm, or from about 0.5 to about 20 mm, or from about 0.5 to about 15 mm, or from about 0.5 to about 10 mm, or from about 0.5 to about 9 mm, or from about 0.5 to about 8 mm,or from about 0.5 to about 7 mm, or from about 0.5 to about 6 mm, or from about 0.5 to about 5 mm, or from about 0.5 to about 4 mm, or from about 0.5 to about 3 mm, or from about 0.5 to about 2 mm, or from about 0.5 to about 1.5 mm, or from about 0.5 to about 1 mm, or from about 1 to about 25 mm, or from about 1 to about 20 mm, or from about 1 to about 15 mm, or from about 1 to about 10 mm, or from about 1 to about 9 mm, or from about 1 to about 8 mm, or from about 1 to about 7 mm, or from about 1 to about 6 mm, or from about 1 to about 5 mm, or from about 1 to about 4 mm, or from about 1 to about 3 mm, or from about 1 to about 2 mm, or from about 1 to about 1.5 mm. The film or sheet may be cut or pre-cut in any size suitable for application to a wound or tissue. The film or sheet may be transparent or opaque. Generally, dehydrated compositions which are dehydrated by lyophilization (freeze-dried) are thicker and opaquer than dehydrated compositions which are dehydrated by drying. Although non-limiting, dried compositions could be considered films whereas lyophilized compositions could be considered sheets.

[0210] The carrier or pharmaceutically acceptable carrier of the compositions may be a film or sheet. Exemplary methods for preparing compositions of films or sheets comprise dehydrating compositions of aqueous gels or hydrogels comprising placental tissue or dehydrated placental tissue. The aqueous gel or hydrogel comprising placental tissue or dehydrated placental tissue may be poured into a suitable mold such as a petri dish prior to dehydration. The aqueous-based gel or hydrogel comprising the placental tissue or dehydrated placental tissue may be dehydrated by any suitable means including but not limited to lyophilizing (freeze-drying), evaporative air-drying (e.g., heat-drying or thermal drying with heated air under gravity convection or forced air convection conditions; or air-drying or drying under ambient or room temperature conditions under gravity convection or forced air convection conditions), indirect drying (e.g., vacuum drying), contact drying (e.g., drying through contact with a heated material or source), dielectric drying (e.g., use of radiofrequency or microwaves), infrared drying, and / or supercritical drying. The resultant film or sheet from evaporative air-drying may be referred to as a xerogel. Evaporative air drying may be accomplished under gravity convection or forced air convention conditions at ambient or room temperature or higher temperatures. Drying using heated air may be accomplished in a heating chamber at temperatures above room temperature with gravity convection or with forced air convection conditions. Drying under gravity convection at ambient or room temperature may be accomplished in a chamber or in an open air space such as on a table or bench. In some aspects, the evaporative air-drying is thermal evaporative air-drying with heated air under gravity convection or forced air convection conditions. In other aspects, the evaporative air-drying is ambient or room temperature evaporative air-drying under gravity convection or forced air convection conditions. In still other aspects, the evaporative air-drying is room temperature evaporative air-drying under gravity convection conditions. Lyophilizing, or freeze-drying, may be accomplished by first freezing the aqueous gel or hydrogel in a freezer, e.g., at -80 °C, then drying in a lyophilizer; or by freezing and drying in a lyophilizer. In some aspects the gel or hydrogel comprising placental tissue or dehydrated placental tissue is dehydrated by evaporative air-drying or lyophilization. The film or sheet is considered “dehydrated” or “dry” when it has a water content (moisture content) of less than 15% w / w, or less than 14% w / w, or less than 13% w / w, or less than 12% w / w, or less than 10% w / w, or less than 9% w / w, or less than 8% w / w, or less than 7% w / w, or less than 6% w / w, or less than 5% w / w, or less than 4% w / w, or less than 3% w / w, or less than 2% w / w, less than 1% w / w, or less than 0.5% w / w. In some aspects the water content is less than 5% w / w, or less than 4% w / w, or less than 3% w / w. The dehydrated film or sheet may, for example, have a water content of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14% w / w, or any percentage derivable therein. The water content (moisture content) of the dehydrated film or sheet may be determined by methods known to one of skill in the art such as by Karl Fischer titration or by oven drying. For example, the dehydrated film or sheet may be placed in a 65 °C oven for 3 minutes at atmospheric pressure and measuring the weight loss after incubation to determine moisture content.

[0211] The placental tissue pieces or particulate, cryopreserved placental tissue pieces or particulate, dehydrated placental tissue pieces or particulate, dried placental tissue pieces or particulate, or lyophilized placental tissue pieces or particulate is uniformly dispersed within the aqueous gel or hydrogel carrier prior to the dehydration step to form the dehydrated film or sheet. A rheology modifier, such as a gellant, may be used to form the aqueous gel or hydrogel prior to dehydration by the addition of the rheology modifier to water or an aqueous medium. The placental tissue pieces or particulate may be added prior to or after the addition of the rheology modifier. The placental tissue pieces or particulate may first be suspended in water or a buffer solution, such as PBS, prior to its addition to wet the tissue to form a slurry then the slurry may further be homogenized with a homogenizer such as a Tissue Tearor™ to facilitate incorporation of the tissue into the aqueous gel or hydrogel carrier. Mixing of the placental tissue pieces or particulate and rheology modifier in the water or aqueous medium may be conducted using methods known to one of skill in the art using suitable mixing equipment known to one of skill in the art, such as propeller mixers, dissolvers, homogenizers, and the like. Suitable rheology modifiers include, but are not limited to cellulose ethers,microcrystalline cellulose, acrylic polymers, alginates, gums, and organoclays, examples of which may be found in “Rheology Modifiers Handbook, Practical Use and Application,” William Andrew Publishing, 2000, herein incorporated by reference. In some aspects, the carrier comprises one or more rheology modifiers. In some aspects, the rheology modifier is a cellulose ether. In some aspects, the carrier comprises one or more cellulose ethers.

[0212] Cellulose ethers include non-ionic and anionic cellulose ethers and are available in a variety of viscosity grades. Nonionic cellulose ethers are high-molecular-weight compounds that may be made by replacing the hydrogen atoms of hydroxyl groups in the glucose units of cellulose with alkyl or hydroxylalkyl groups. Non-limiting examples of non-ionic alkyl cellulose ethers include methyl cellulose (MC), ethyl cellulose (EC), and ethyl methyl cellulose (EMC). Non-limiting examples of non-ionic hydroxyalkyl cellulose ethers include hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxymethyl cellulose (HMC), hydroxypropylmethyl cellulose (HPMC), ethylhydroxyethyl cellulose (EHEC), hydroxyethylmethy cellulose (HEMC), methylhydroxyethyl cellulose (MHEC), methylhydroxypropylcellulose (MHPC), and hydroxyethylcarboxymethyl cellulose (HECMC). There are a wide range of commercial sources for each of these cellulose ethers (e.g., Dow Chemical Company, Ashland, Spectrum Chemical). HEC is available under the name NATROSOL™ from Ashland. HPC is available under the name KLUCEL™ from Ashland. HPMC (also known as 64ypromellose) is available under the name BENECEL™ from Ashland. An example of an anionic cellulose ether is sodium carboxymethyl cellulose (CMC) available commercially under the name AQUALON™ from Ashland. The cellulose ethers are available in cosmetic grades and pharmaceutical grades (USP / NF) and are suitable for use in the disclosed compositions. The concentration of the cellulose ether in the composition may vary as needed to achieve a particular characteristic of the dehydrated film or sheet. The concentration of the cellulose ether in the composition (cumulative concentration if more than one cellulose ether is present) may be from about 20 % to about 80% w / w, or from about 30% to about 80% w / w, or from about 40% to about 80% w / w for a composition in the dehydrated state. The carrier may comprise one or more cellulose ethers. In some aspects, the carrier comprises one or more cellulose ethers. In some aspects, the cellulose ether is a non- ionic cellulose ether. In some aspects, the non-ionic cellulose ether is hydroxyethyl cellulose (HEC) or hydroxypropyl cellulose (HPC), or mixtures thereof.

[0213] The carrier may further comprise one or more plasticizers, which may serve to enhance the desired physical properties, such as brittleness and flexibility, of dehydrated films or sheets. Non-limiting examples of plasticizers include hydrophilic polyols, hydrophilicpolymeric polyols, propylene glycol, glycerol, polyethylene glycol (PEG), polypropylene glycol, poloxamers, and povidone. The concentration of the plasticizer in the composition may be from about 0% to about 30% w / w, or from about 5% to about 30% w / w, or from about 10% to about 25% w / w for the composition in the dehydrated state. In some aspects, the plasticizer is a hydrophilic polyol. Suitable hydrophilic polyols are water-soluble, polar aliphatic alcohols with at least two hydroxyl groups, and may include polymeric polyols, e.g., polyethylene glycols and poloxamers. In some aspects, the hydrophilic polyol is a hydrophilic polymeric polyol. In some aspects, the hydrophilic polymeric polyol is one or more polyethylene glycols. In some aspects, the polyethylene glycol is polyethylene glycol 600.

[0214] Polyethylene glycols (PEG) are homo-polymers of ethylene glycol and water represented by the formula H(OCH2CH2)nOH, in which n represents the average number of oxy ethylene groups. Polyethylene glycols may be either liquid or solid at 25 °C. depending on their molecular weights. The following non-limiting examples are suitable for use with the disclosed compositions and are described using U.S. Pharmacopeial Convention (USP) nomenclature: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, and polyethylene glycol 600. The following non-limiting examples of solid polyethylene glycols are suitable for use with the disclosed compositions and are described using USP nomenclature: polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1450, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3250, polyethylene glycol 3350, polyethylene glycol 3750, polyethylene glycol 4000, polyethylene glycol 4250, polyethylene glycol 4500, polyethylene glycol 4750, polyethylene glycol 5000, polyethylene glycol 5500, polyethylene glycol 6000, polyethylene glycol 6500, polyethylene glycol 7000, polyethylene glycol 7500, and polyethylene glycol 8000. Such liquid and solid polyethylene glycols are available commercially from the DOW Chemical Company under the CARBOWAX™ and SENTRY™ names and from the BASF Corporation under the PLURACARE® and PLURIOL® names. Polyethylene glycols are available in cosmetic grades and pharmaceutical grades (USP / NF) and are suitable for use in the disclosed compositions. The concentration of the polyethylene glycol in the composition (cumulativeconcentration if more than one polyethylene glycol is present) may be from about 0% to about 30% w / w, or from about 5% to about 30% w / w, or from about 10% to about 25% w / w for the composition in the dehydrated state. In some aspects, the carrier comprises polyethylene glycol (PEG) 600. PEG 600 is available under the name PLURACARE® E 600 from BASF and under the name CARBOWAX™ SENTRY™ Polyethylene Glycol 600 NF from Dow.

[0215] The concentration of placental tissue as pieces or particulate, cryopreserved placental tissue pieces or particulate, dehydrated placental tissue as pieces or particulate, dried placental tissue pieces or particulate, or lyophilized placental tissue pieces or particulate in the film or sheet composition may be an amount effective to provide an environment to supporting the wound healing process by allowing cellular migration, vascular ingrowth, and the formation of granulation tissues when the composition is applied to a wound or damaged tissue as demonstrated by the examples disclosed herein. The concentration of placental tissue pieces or particulate, cryopreserved placental tissue pieces or particulate, dehydrated placental tissue pieces or particulate, dried placental tissue pieces or particulate, or lyophilized placental tissue pieces or particulate in the dehydrated film or sheet composition may be from about 1% to about 75% w / w, or from about 1% to about 60% w / w, or from about 1% to about 50% w / w, or from about 2% to about 50% w / w, or from about 3% to about 50% w / w, or from about 4% to about 50% w / w, or from about 5% to about 50% w / w, or from about 6% to about 50% w / w, or from about 7% to about 50% w / w, or from about 8% to about 50% w / w, or from about 9% to about 50% w / w, or from about 10% to about 50% w / w, or from about 1% to about 40% w / w, or from about 2% to about 40% w / w, or from about 3% to about 40% w / w, or from about 4% to about 40% w / w, or from about 5% to about 40% w / w, or from about 6% to about 40% w / w, or from about 7% to about 40% w / w, or from about 8% to about 40% w / w, or from about 9% to about 40% w / w, or from about 10% to about 40% w / w, of the total composition for compositions in the cryopreserved or dehydrated state. For example, the concentration of the placental pieces or particulate, cryopreserved placental tissue pieces or particulate, dehydrated placental tissue pieces or particulate, dried placental tissue pieces or particulate, or lyophilized placental tissue pieces or particulate in the film or sheet composition may be about 1%, or about 2%, or about 3%, about 4% or about 5%, or about 10%, or about 11%, or about 15%, or about 20%, or about 25%, or about 30%, or about 39%, or about 40%, or about 42%, or about 45%, or about 50%, or about 55%, or about 60% or about 65%, or about 70%, or about 75% w / w. In some aspects, the cryopreserved placental tissue, dehydrated placental tissue, dried placental tissue, or lyophilized placental tissue may be in particulate form.

[0216] In some aspects, the cryopreserved placental tissue, dehydrated placental tissue, dried placental tissue, or lyophilized placental tissue may be in foam form.

[0217] In some aspects, preparation of film form products comprises utilization of raw material that comprises particulate tissue, which is processed by both lyophilization and by drying, sequentially. In some aspects, film form products can be the thinnest (e.g., can be less than 0.5 mm) of the various product types (e.g., film, foam, and sheet). In some aspects, film form products can appear to the eye like a semi-clear paper.

[0218] In some aspects, preparation of sheet form products comprises utilization of raw material that can be tissue that has been cut open (e.g., cut-open placental tissues), which can be processed by lyophilization. In some aspects, preparation of a sheet form product provides products that are at least, at most, exactly, or between any two of 0.5 to 2 mm thick. In some aspects, sheet form products are thicker than film form products. In some aspects, sheet form products are denser (e.g., lower tissue porosity) than film form products. In some aspects, sheet form products can appear to the eye like a semi-clear paper. In some aspects, a sheet form product can be processed by drying. In some aspects, a sheet form product processed by drying may be thinner than a sheet form product processed by lyophilization.

[0219] In some aspects, preparation of a foam form products comprises utilization of particulate tissue (e.g., placental tissues which has been processed into particles), which can be processed by lyophilization. In some aspects, the thickness of a foam form product is controllable depending on loading volume before lyophilization. In some aspects, a foam form product has higher tissue porosity when compared to sheet form or film form products, and can appear to the eye to comprise a sponge shape and / or consistency.

[0220] Disclosed is a non-limiting exemplary method of a method of making a dehydrated composition comprising dehydrated placental tissue, the method comprising:

[0221] (a) providing dehydrated placental tissue;

[0222] (b) providing an aqueous based pharmaceutically acceptable carrier;

[0223] (c) combining (a) and (b) and mixing until uniform; and

[0224] (d) dehydrating the resultant mixture, thereby forming the dehydrated composition;

[0225] wherein the aqueous based carrier has a water content of at least 50% w / w, wherein the dehydrated composition is a film or sheet, and wherein the film or sheet has a water content of less than 5% w / w or less than 3% w / w. In some aspects, the dehydrating step (d) is conducted by drying or lyophilization.

[0226] Disclosed is a non-limiting exemplary dehydrated composition comprising:

[0227] (a) dehydrated placental tissue; and

[0228] (b) an aqueous based pharmaceutically acceptable carrier comprising:

[0229] (i) one or more cellulose ethers, and

[0230] (ii) one or more plasticizers,

[0231] wherein the aqueous based carrier has a water content of at least 50% w / w prior to dehydration of the composition, wherein the composition is dehydrated by drying or lyophilization, wherein the dehydrated composition is a film or sheet, and wherein the film or sheet has a water content of less than 5% w / w or less than 3% w / w. In some aspects, the one or more cellulose ethers comprise hydroxyethyl cellulose (HEC) or hydroxypropyl cellulose (HPC), or mixtures thereof. In some aspects, the one or more plasticizers comprise one or more polyethylene glycols. In some aspects, the one or more polyethylene glycols comprise polyethylene glycol 600.

[0232] An exemplary feature of the dehydrated film and sheet compositions disclosed herein is that they have the capability of forming a hydrogel when they come in contact with water or other aqueous medium, such as a buffer or saline solution, or moisture from the wound itself. Thus, when the dehydrated film or sheet composition is applied to a wound, the moisture from the wound or external source reconstitutes the composition back into a hydrogel. The resultant hydrogel may have a sufficient viscosity that it remains on the wound and does not drain off. The dehydrated film or sheet may be capable of not disintegrating as is the case with other film wound dressings, but rather forms a hydrogel that remains on the wound creating an environment and a physical matrix or scaffold that provides conditions to support the wound healing process by allowing cellular migration, vascular ingrowth, and the formation of granulation tissues. The amount of the cellulose ether and / or plasticizer within the dehydrated film or sheet composition may vary as needed to achieve a desired viscosity in the resultant hydrogel that is formed when the dehydrated film or sheet comes in contact with water or other aqueous medium. In some aspects, the resultant hydrogel has a sufficient viscosity so that it will remain in a wound bed and not drain off the wound. The viscosity of the resultant hydrogel may be measured using a viscometer, such as a Brookfield viscometer. A preferred method of measuring the viscosity of the resultant hydrogel is using a Brookfield viscometer Model RV- DV2T cone & plate viscometer using cone spindle CP-52 at 0.5 RPM at RT and running for 1 minute. The sample for the viscosity measurement may be prepared by contacting about 1 part by weight of the dehydrated film or sheet with about 20 parts of water or other aqueous medium such as normal saline solution. Viscosity values may be reported in centipoise (cps). Viscosity values using the preferred method may be from about 100 cps to about 100,000 cps, or from about 100 cps to about 75,000 cps, or from about 100 cps to about 50,000 cps, or from about100 cps to about 40,000 cps, of from about 100 cps to about 30,000 cps, or from about 100 cps to about 25,000 cps, or from about 100 cps to about 20,000 cps, or from about 100 cps to about 19,000 cps, or from about 100 cps to about 18,000 cps, or from about 100 cps to about 17,000 cps, or from about 100 cps to about 16,000 cps, or from about 100 cps to about 15,000 cps, or from about 100 cps to about 14,000 cps, or from about 100 cps, to about 13,000 cps, or from about 100 cps to about 12,000 cps, or from about 500 cps to about 100,000 cps, or from about 500 cps to about 75,000 cps, or from about 500 cps to about 50,000 cps, or from about 500 cps to about 40,000 cps, of from about 500 cps to about 30,000 cps, or from about 500 cps to about 25,000 cps, or from about 500 cps to about 20,000 cps, or from about 500 cps to about 19,000 cps, or from about 500 cps to about 18,000 cps, or from about 500 cps to about 17,000 cps, or from about 500 cps to about 16,000 cps, or from about 500 cps to about 15,000 cps, or from about 500 cps to about 14,000 cps, or from about 500 cps, to about 13,000 cps, or from about 500 cps to about 12,000 cps, or from about 1000 cps to about 100,000 cps, or from about 1000 cps to about 75,000 cps, or from about 1000 cps to about 50,000 cps, or from about 1000 cps to about 40,000 cps, of from about 1000 cps to about 30,000 cps, or from about 1000 cps to about 25,000 cps, or from about 1000 cps to about 20,000 cps, or from about 1000 cps to about 19,000 cps, or from about 1000 cps to about 18,000 cps, or from about 1000 cps to about 17,000 cps, or from about 1000 cps to about 16,000 cps, or from about 1000 cps to about 15,000 cps, or from about 1000 cps to about 14,000 cps, or from about 1000 cps, to about 13,000 cps, or from about 1000 cps to about 12,000 cps, or from about 5000 cps to about 100,000 cps, or from about 5000 cps to about 75,000 cps, or from about 5000 cps to about 50,000 cps, or from about 5000 cps to about 40,000 cps, of from about 5000 cps to about 30,000 cps, or from about 5000 cps to about 25,000 cps, or from about 5000 cps to about 20,000 cps, or from about 5000 cps to about 19,000 cps, or from about 5000 cps to about 18,000 cps, or from about 5000 cps to about 17,000 cps, or from about 5000 cps to about 16,000 cps, or from about 5000 cps to about 15,000 cps, or from about 5000 cps to about 14,000 cps, or from about 5000 cps, to about 13,000 cps, or from about 5000 cps to about 12,000 cps, or from about 10,000 cps to about 100,000 cps, or from about 10,000 cps to about 75,000 cps, or from about 10,000 cps to about 50,000 cps, or from about 10,000 cps to about 40,000 cps, of from about 10,000 cps to about30,000 cps, or from about 10,000 cps to about 25,000 cps, or from about 10,000 cps to about20,000 cps, or from about 10,000 cps to about 19,000 cps, or from about 10,000 cps to about18,000 cps, or from about 10,000 cps to about 17,000 cps, or from about 10,000 cps to about16,000 cps, or from about 10,000 cps to about 15,000 cps, or from about 10,000 cps to about14,000 cps, or from about 10,000 cps, to about 13,000 cps, or from about 10,000 cps to about12,000 cps, or from about 12,000 cps to about 100,000 cps, or from about 12,000 cps to about 75,000 cps, or from about 12,000 cps to about 50,000 cps, or from about 12,000 cps to about40,000 cps, of from about 12,000 cps to about 30,000 cps, or from about 12,000 cps to about25,000 cps, or from about 12,000 cps to about 20,000 cps, or from about 12,000 cps to about19,000 cps, or from about 12,000 cps to about 18,000 cps, or from about 12,000 cps to about17,000 cps, or from about 12,000 cps to about 16,000 cps, or from about 12,000 cps to about15,000 cps, or from about 12,000 cps to about 14,000 cps, or from about 12,000 cps, to about13,000 cps or from about 15,000 cps to about 100,000 cps, or from about 15,000 cps to about 75,000 cps, or from about 15,000 cps to about 50,000 cps, or from about 15,000 cps to about40,000 cps, of from about 15,000 cps to about 30,000 cps, or from about 15,000 cps to about25,000 cps, or from about 15,000 cps to about 20,000 cps, or from about 20,000 cps to about100,000 cps, or from about 20,000 cps to about 75,000 cps, or from about 20,000 cps to about 50,000 cps, or from about 20,000 cps to about 40,000 cps, of from about 20,000 cps to about 30,000 cps, or from about 20,000 cps to about 25,000 cps.B. EXCIPIENTS AND ADDITIONAL INGREDIENTS

[0233] The compositions disclosed herein may further comprise excipients, functional ingredients, and additional ingredients. Non-limiting examples of such ingredients include active pharmaceutical ingredients (APIs), absorbents, antimicrobial agents, antioxidants, antibiofilm agents, binders, buffering agents (e.g., Tris buffer solutions and PBS), bulking agents, chelating agents, colorants, debriding agents, dyes, biocides, deodorant agents, emulsion stabilizers, film formers, fragrance ingredients, humectants, gellants (e.g., cellulose ethers, microcrystalline cellulose, acrylic polymers, alginates, gums, organoclays), lytic agents, enzymes, proteolytic enzymes, opacifying agents, oxidizing agents, pH adjusters, plasticizers, preservatives (e.g., methylparaben, propylparaben, benzyl alcohol), reducing agents, emollients, humectants, hydrophilic polyols, hydrophilic polymeric polyols, polyethylene glycols, moisturizers, surfactants, emulsifying agents, cleansing agents, foaming agents, hydrotopes, solvents, suspending agents, rheology modifiers, viscosity control agents, viscosity increasing agents (e.g., thickeners), vulnerary agents, and propellants. In some aspects, the composition does not include or contain an enzyme, a proteolytic enzyme, an antibiofilm agent, and / or a debriding agent. In some aspects, the composition does not include or contain an active pharmaceutical ingredient.VI. PLACENTAL TISSUE DEVICES

[0234] In another aspect, the placental tissues or compositions thereof disclosed herein may be devices, including wound dressings comprising native viable cells, native therapeutic factors, extracellular matrix (ECM) components, cytokines, growth factors, or any combination thereof. In some aspects, the device may be a single use sterile dressing intended for the management of wounds (e.g., chronic, acute, and post-surgical exuding wounds) by providing a topical wound covering which functions as a protective barrier for the wound. In some aspects, the device may be engineered to provide a biodegradable and / or terminally sterilized, ECM wound covering that is derived from human placental tissue. In some aspects, the device may comprise a matrix of collagens, proteoglycans, ECM glycoproteins, or any combination thereof, and may have a tensile strength and thickness to be sutured onto the wound bed. In some aspects, the device may comprise a matrix of collagens, proteoglycans, and ECM glycoproteins derived from human placental tissue with an anticipated wear time of 10-21 days (e.g., at least, at most, exactly, or between any two of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days) before it is either (1) resorbed by the body and use of the device is discontinued or (2) the device is replaced with a new device for upwards of ten applications for management of the hardest to heal wounds.

[0235] In some aspects, the device may be supplied in a variety of sterile sheet configurations which are packaged in double peel-open packages and are intended for singleuse only. In some aspects, the device may be terminally sterilized using electron beam (E- beam), irradiation, gamma irradiation, supercritical CO2 sterilization, and / or ethylene oxide (EtO) sterilization.

[0236] In some aspects, the device may be used by a licensed healthcare practitioner. In some aspects, the device may be used for management of chronic, acute, and post-surgical exuding wounds including partial- and full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled, surgical wounds (e.g., donor sites / grafts, post-Mohs surgery, post-laser surgery, podiatric, wound dehiscence), trauma wounds (e.g., abrasions, lacerations, second-degree burns, and skin tears), and draining wounds, optionally wherein the device may be a biodegradable and / or terminally sterilized, wound covering that is derived from human placental tissue.

[0237] In another aspect, the device may be manufactured in different dimensions and / or sizes. The device may be, for example 1 to 10 cm wide (e.g., at least, at most, exactly, or between any two of 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm wide), 1 to 10 cm long (e.g., at least, at most, exactly, or between any two of 1 cm, 2 cm, 3 cm, 4 cm, 5cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm long), and 0.1 to 5 mm thick (e.g., at least, at most, exactly, or between any two of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, or 5 mm thick). The wound dressing type of device may be manufactured in different sizes of sheets, including the exemplary sizes in Table 1.Table 2Exemplary Placental Product Dimensions

[0238] In some aspects, a surface of the wound dressing may not have a required orientation when placed on the wound and may be compositionally equivalent (e.g., either side of the wound dressing may be placed on the wound surface).

[0239] In some aspects, the device may comprise native viable cells. In some aspects, the device does not comprise viable cells. In some aspects, the device is not decellularized. In some aspects, the device is not decellularized but does not comprise viable cells. In some aspects, the device is decellularized. In some aspects, the device comprises native viable cells, native therapeutic factors, extracellular matrix (ECM) components, cytokines, growth factors, or any combination thereof. In some aspects, the device comprises a matrix of collagens, proteoglycans, and ECM glycoproteins derived from human placental tissue. Extracellular matrix components of placental tissue can include, but are not limited to, collagen, aggrecan, agrin, chondroitin sulfate proteoglycans, elastin, fibrinogen, fibrillin, fibronectin, ficolin-2, heparin sulfate proteoglycan, hyaluronic acid, dermatan sulfate, chondroitin sulfate, laminins, nidogen, vitronectin, osteopontin, or a combination thereof. The device may further comprise growth factors and / or cytokines derived from human placental tissue. Growth factors and / or cytokines can include, but are not limited to, TIMP-1, TIMP-2, TIMP-4, IFN y, lactoferrin, HBD, HNP-3, elafin / trappin-2, SLP1, TGF-p, TGF-a, activin, VEGF, P1GF, FGF, EGF, HGF,IGF-1, PDGF, adiponectin, IGFBP, MMP-2, MMP-9, or a combination thereof. Growth factors and / or cytokines may be included in the device in an amount that is 0.001% to 99% of the device components, e.g., at least, at most, exactly, or between any two of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0240] In another aspect, the device may comprise residual deoxyribonucleic acid (DNA) at a level that is equivalent or lower than other animal derived tissue-based products cleared by the United States Food and Drug Administration or equivalent regulatory bodies in other jurisdictions.

[0241] In another aspect, the device may comprise materials derived from human placental tissue that may be used to provide and support the physical structure of the dressing. In some aspects, the device may comprise materials derived from human placental tissue that are not used for biological actions related to wound healing (e.g., to accelerate wound healing). In some aspects, the device may not comprise antimicrobials, drugs, or biologies. In some aspects, the device may comprise metabolically active, viable cells from the placental tissue after being processed according to the methods disclosed herein. In some aspects, the device may not comprise metabolically active cells from the placental tissue after being processed according to the methods disclosed herein.VII. METHODS OF USE

[0242] Disclosed are methods of treating a wound or tissue defect comprising administering a placental tissue to the wound or tissue defect. Disclosed are methods of treating a wound or tissue defect comprising administering one or more of the placental tissues disclosed herein to the wound or tissue defect.

[0243] The compositions disclosed herein can, in some aspects, be useful for the treatment of wounds of a subject by applying the compositions to or on the wound. A wound can include a disruption of the structure and function of tissue. In addition to the other non-limitingexamples disclosed elsewhere herein, wounds can include: internal organ wounds; mucous membrane wounds; vascular tissue wounds; soft tissue wounds including ligaments, tendons, and cartilage; bone wounds; and dermal wounds. In some aspects, the wound is a dermal wound. In some aspects, the composition is applied topically to a dermal wound. In some aspects, the composition after application is in direct contact with at least a portion of the wound surface.

[0244] A dermal wound may involve the disruption of the skin and associated soft tissue architecture. Dermal wounds may be partial or full thickness wounds. They may also be acute wounds, chronic wounds, or bums, which may be acute or chronic. Non-limiting examples of a bum wound include a superficial (first degree) bum, a partial thickness (second degree) bum, a full thickness (third degree) burn, or a radiation bum. Non-limiting examples of a chronic wound include a dermal ulcer, a diabetic ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, an arterial ulcer, an arterial leg ulcer, a decubitus ulcer, a stasis ulcer, an ischemic ulcer, a vascular ulcer, a pressure ulcer (stage I-IV), a podiatric wound, a draining wound, a tunneling wound, or an undermining wound. Non-limiting examples of an acute wound include a trauma wound, a laceration, an abrasion, a skin tear, a skin lesion, a blister, a surgical incision, a donor skin site, a skin graft, a laser surgery wound, a Mohs surgery wound, or a dehisced wound. In some aspects, the dermal wound includes necrotic tissue. In other aspects, the dermal wound does not contain necrotic tissue.

[0245] In some aspects, compositions disclosed herein may provide a topical wound covering which functions as a protective barrier for the wound.

[0246] In some aspects, compositions disclosed herein may be used as a dressing to provide a physical barrier for the management of chronic wounds (such as diabetic foot ulcers, venous leg ulcers, pressure ulcers), acute wounds, and in the post-operative care of surgical incisions. In some aspects, compositions disclosed herein may be sutured onto the wound bed, which either independently or in combination with its physical barrier function, may protect the innate wound healing response. In some aspects, the subject device may also act as a biodegradable scaffold that supports the body’s own wound healing processes.

[0247] In some aspects, the compositions disclosed herein may be used, optionally as a wound dressing, for management of chronic, acute and post-surgical exuding wounds including partial- and full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled, surgical wounds (e.g., donor sites / grafts, post-Mohs surgery, postlaser surgery, podiatric, wound dehiscence), trauma wounds (e.g., abrasions, lacerations, second-degree bums and skin tears), and draining wounds.

[0248] In some aspects, the compositions disclosed herein may be used as a dressing to provide a protective cover to chronic wounds, acute wounds, and in post-operative care of surgical incisions. In some aspects, the dressing may promote an environment that helps with wound management by serving as protective barrier, which may be supported by the collagen composition and structural thickness (e.g., a range of at least, at most, exactly, or between any two of 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm) of the dressing. In some aspects, the dressing’s thickness and tensile strength may enable the dressing to be sutured on the wound.

[0249] In some aspects, the compositions disclosed herein may provide the benefits, including barrier that protects the wound environment, ability to suture the subject device on the wound, terminal sterilization and viral inactivation reducing the risk of microbial and viral contamination of the device, ability of biodegrading within 10-21 days (e.g., at least, at most, exactly, or between any two of 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days), elasticity properties of subject device which may allow it to conform to complex wound anatomy, and / or availability in multiple sizes.

[0250] In another aspect, the compositions disclosed herein may be applied to the wound periodically, for example, daily. A therapeutic regiment could be followed to include periodic dressing changes with wound cleansing and application of fresh composition between changes until the wound is healed. The compositions may be applied in conjunction with the application of other wound dressings including but not limited to gauze bandages, sponge wound dressings, foam wound dressings (e.g., Allevyn™ foam dressing), antimicrobial wound dressings, ECM based wound dressings, placental tissue wound dressings, wound debriding dressings, calcium alginate dressings, hydrogels, and wound dressings with vulnerary agents. For example, after application of the composition, the wound may be covered with another wound dressing. The composition may be applied before or after the application of another wound dressing. In some aspects, the composition comprises lyophilized placental tissue in various forms (e.g., particulate, sheet, and / or foam) and may be applied to the wound before the application of another wound dressing, and in other aspects, the composition may be applied after the application of the other wound dressing. Dehydrated films, sheet, and / or foam compositions may be applied to the wound dry or moistened with an aqueous medium such as saline solution before or after application.

[0251] In some aspects, the compositions disclosed herein may allow for the cellular migration, vascular ingrowth, and / or the formation of granulation tissues when thecompositions are applied to a wound or damaged tissue. Thus, use of the compositions may support and facilitate the wound healing process.VIII. PACKAGING

[0252] The compositions of the present disclosure may be packaged in any package configuration suitable, for example, for use in storing, shipping, and / or using the compositions of the present disclosure. Non-limiting examples of packaging configurations may include containers, such as plastic packages, foil packages, pouches, packets, and / or boxes. In certain aspects where the composition is flowable (e.g., in liquid or hydrogel form), the compositions bottles, jars, bottles with pumps, toddles, tubes (e.g., aluminum, plastic, or laminated), jars, non-aerosol pump sprayers, and / or aerosol containers could be used. The packages may be configured for single-dose or multiple-dose administration.

[0253] Containers such as kits that have multiple compartments may also be used. For instance, a composition of the present disclosure may be in the form of particulate, sheet, and / or foam and be placed in one compartment. A second compartment may include a composition that may include, for example, water or other aqueous solution. This may allow the particulate, sheet, and / or foam to be mixed with the second composition to form a hydrogel. The kit could also include a mixer (e.g., spoon, rod, or paddle) to mix the two compositions and / or an applicator (e.g., spoon, rod, or paddle) to apply the hydrogel to a wound or tissue such as damaged tissue. Kits may also include 3, 4, 5, or more additional compartments or containers.

[0254] In various aspects, the placental tissue and other compositions described herein can be provided in a kit. The kit can also include combinations of the placental tissue, preservation agents, water, saline, or a buffer such as, but not limited to phosphate buffered saline (PBS), in a solution comprising a stabilizing agent such as, but not limited to bovine serum albumin (BSA), Plasma-Lyte A or other clinically available electrolyte solutions, with human bodily fluids or a combination thereof described herein.

[0255] In one aspect, disclosed are kits comprising a disclosed placental tissue and one or more of: (a) water, saline, or a buffer such as, but not limited to phosphate buffered saline (PBS), in a solution comprising a stabilizing agent such as, but not limited to bovine serum albumin (BSA), Plasma-Lyte A or other clinically available electrolyte solutions, with human bodily fluids or a combination thereof; and (b) instructions for thawing or reconstituting the tissue.

[0256] In various aspects, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a fragrance or other cosmetic ingredient. In suchaspects, the kit can include instructions for the placental tissue and the other ingredients, or for using one or more compounds together with the other ingredients.

[0257] Packaging may also include informational material relating to the compositions of the present disclosure. In various aspects, the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or to the use of the placental tissue for the methods described herein. Instructions may include an explanation of how to apply, use, and maintain the products or compositions.EXAMPLES

[0258] The following examples are included to demonstrate aspects of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.I. EXAMPLE 1 - DEHYDRATED PLACENTAL TISSUE DRESSINGS

[0259] Dehydrated placental tissue dressings may be produced in a particulate form, in a sheet form, and / or in a foam form.

[0260] Sheet form dehydrated placental tissue dressings consisting of double amnion layers were manufactured by (a) obtaining one or more amniotic tissues, each amniotic tissue having a stromal layer opposite an epithelial layer, and forming a amniotic tissue laminate by (1) folding one of the one or more amniotic tissues to contact the epithelial layer of a first portion of the amniotic tissue with the epithelial layer of a second portion of the amniotic tissue; or (2) layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the epithelial layer of the first of the amniotic tissues with the epithelial layer of the second of the amniotic tissues. The amniotic tissue laminates were then lyophilized or air-dried.

[0261] FIG. 1 depicts exemplary lyophilized (left) or air-dried (right) placental tissue dressings in a sheet form. FIG. 2 depicts a single layer of amniotic tissue 10a used to produce a layered amniotic tissue laminate 10b. The single layer amniotic tissue 10a includes a single epithelial layer I la and a single stromal layer 11b. The amniotic tissue laminate 10b includes two layers or sheets of amniotic tissue 10a, each layer or sheet of amniotic tissue 10a having a single epithelial layer I la and a single stromal layer 11b. The appearance of the amnionlaminate after lyophilization included unclear surface color that is similar to original tissue (FIG. 1, left). The appearance of the amnion laminate after air-drying showed a clear surface color (FIG. 1, right). Additionally, as shown in FIG. 2, the thickness of the amnion laminate increases when multiple layers of amniotic tissues are combined, but roughness and porosity of the amniotic tissues does not substantially increase or decrease.

[0262] The finished prototypes were visually assessed to check for cracks, holes, color, and delamination. After rehydration of the randomly selected prototypes with saline, units were investigated whether the units were delaminated by 1 -minute rehydration. All major and minor issues were noted for each prototype, and any major issues were compared as %. Delamination, cracking, and holes were identified as “major” issues, and mismatching of the layer to the layer and color were identified as “minor” issues.

[0263] From 6 donors, 93 total units of double-layer amnion facing stromal layer to stromal layer and 29 units of double-layer amnion facing epithelial layer to epithelial layer were processed by lyophilization, and 3 units of double-layer amnion facing stromal layer to stromal layer were processed by heat-drying. As shown in Table 3, the amnion laminates prepared according to the methods disclosed herein (i.e., lyophilization of amnion coupled stromal layer to stromal layer) did not experience any major issues (i.e., delamination) before reconstitution or rehydration and less than 3% of the laminates experienced major issues (i.e., delamination) after reconstitution or rehydration. Seven units experienced minor issues before rehydration (FIG. 3, left). Two units, or 2.47% of units, experienced major issues after rehydration (FIG. 4, left).Table 3 Stromal-to-Stromal Lyophilized Double Layer Amnion

[0264] As shown in Table 4, 3.4% of units of lyophilized amnion coupled epithelial layer to epithelial layer experienced major issues before rehydration, and more than 50% of units experienced major issues after rehydration. One unit, or 3.4% of units, experienced major issues before rehydration (FIG. 5, left; FIG. 6, left). Five units, or 55.6% of units, experienced major issues after rehydration (FIG. 6, right).Table 4 Epithelial-to-Epithelial Lyophilized Double Layer Amnion

[0265] As shown in Table 5, no units of heat-dried amnion coupled stromal layer to stromal layer experienced major or minor issues before or after rehydration.Table 5 Stromal-to-Stromal Heat-Dried Double Layer Amnion

[0266] Both lyophilization and heat-drying the amnion laminates yields dressings having similar thicknesses and elasticity that are less prone to delamination. Lyophilized amnion laminates, which can retain protein stability and growth factors, may be ideal for wound protection and healing purposes, for instance, by providing anti-inflammation and antibacterial effects, or by acting as a barrier or supporting epithelium. In some aspects, lyophilized double-amnion may be suitable as a wound cover. Heat-dried amnion laminates, in which collagen-based tissue structures may be thermally cross-linked, can provide a durable structure that can support wrapping or other application for treatment of tissue injuries and can withstand various physical and mechanical challenges; it may also provide an anti-adhesion function. In some aspects, heat-dried double-amnion may be suitable as a tendon or ligament wrap. Processing amnion by folding may be, in some aspects, an easier processing technique than combining two separated pieces of amnion.* * *

[0267] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS1. A method of manufacturing an amniotic tissue graft, the method comprising: obtaining one or more amniotic tissues, each amniotic tissue comprising a stromal layer and an epithelial layer opposite the stromal layer; and forming an amniotic tissue laminate by: folding one of the one or more amniotic tissues to contact the stromal layer of a first portion of the amniotic tissue with the stromal layer of a second portion of the amniotic tissue; or layering a first of the amniotic tissues with a second of the amniotic tissues by contacting the stromal layer of the first of the amniotic tissues with the stromal layer of the second of the amniotic tissues.

2. The method of claim 1, further comprising inserting or layering a reinforcing material between the stromal layers of the amniotic tissue(s).

3. The method of claim 2, wherein the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or biosynthetic material.

4. The method of any one of claims 1-3, further comprising lyophilizing the amniotic tissue laminate.

5. The method of claim 4, wherein the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for wound treatment.

6. The method of claim 5, wherein the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a bum, a thermal burn, a chemical burn, a partial thickness bum, or a full thickness bum; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum.

7. The method of claim 1, further comprising air-drying the amniotic tissue laminate.

8. The method of claim 7, wherein the amniotic tissue graft comprises a surface roughness and tissue porosity suitable for soft tissue in need of repair.

9. The method of claim 8, wherein the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint.

10. The method of any one of claims 1-9, further comprising sterilizing the amniotic tissue laminate.

11. An amniotic tissue graft comprising a lyophilized amniotic tissue laminate, the lyophilized amniotic tissue laminate comprising: an amniotic tissue that has been folded to contact a stromal layer of a first portion of the amniotic tissue with a stromal layer of a second portion of the amniotic tissue; or a first amniotic tissue layered with a second amniotic tissue by contacting a stromal layer of the first amniotic tissue with a stromal layer of the second amniotic tissue.

12. The amniotic tissue graft of claim 11, further comprising a reinforcing material inserted or layered between the stromal layers of the amniotic tissue(s).

13. The amniotic tissue graft of claim 12, wherein the reinforcing material comprises a xenograft, allograft, ECM-based tissue, porcine small intestinal submucosa, collagen, or synthetic or bio-synthetic material.

14. The amniotic tissue graft of any one of claims 11-13, wherein the amniotic tissue laminate is sterilized.

15. The amniotic tissue graft of any one of claims 11-14, wherein the amniotic tissue laminate is lyophilized.

16. The amniotic tissue graft of any one of claims 11-14, wherein the amniotic tissue laminate is air-dried.

17. A method of treating a wound, the method comprising topically applying to the wound an amniotic tissue graft prepared according to the method of any one of claims 1-10 or the lyophilized amniotic tissue graft of claim 15.

18. The method of claim 17, wherein the wound is: a chronic wound, an ulcer, a diabetic ulcer, a foot ulcer, a diabetic foot ulcer, a venous ulcer, a venous leg ulcer, a leg ulcer, a pressure ulcer, a bum, a thermal burn, a chemical burn, a partial thickness bum, or a full thickness bum; or a laceration, a scrape, an incision, a puncture, a wound caused by a projectile, an epidermal wound, a dermal wound, a surgical wound, an acute wound, a congenital wound, a toxic epidermal necrolysis, an epidermolysis bullosa, or a pyoderma gangrenosum.

19. A method of repairing soft tissue, the method comprising applying to soft tissue in need of repair an amniotic tissue graft prepared according to the method of any one of claims 1-10 or the air-dried amniotic tissue graft of claim 16.

20. The method of claim 19, wherein the soft tissue in need of repair is skin, Achilles tendon, tibialis tendon, patellar tendon, gracilis tendon, anterior cruciate ligament (ACL), posterior cmciate ligament (PCL), collateral ligament, rotator cuff, or tendons in the knee joint or shoulder joint.

Images