Skin graft compositions and methods of making and using same

Abstract

The present invention provides a method of preparing a skin graft composition, comprising: (a) obtaining a skin sample from a subject in need of a skin layer patch or dressing; (b) placing the skin sample in a container with a sterile medium; (c) isolating fibroblasts and keratinocytes from the skin sample to obtain a solution containing fibroblasts and keratinocytes or reshaping and resizing the skin sample to obtain a solution containing a reshaped and resized skin sample; and (d) combining the fibroblasts and keratinocytes with a matrix to form a skin graft composition. The present invention also provides a method of providing a skin graft composition to a subject, and a method of providing a skin layer patch or dressing, or a skin graft to a subject in need of a skin graft composition.

Description

[Background technology]

[0001] 2. Background of the Invention Loss of skin tissue due to burns, injury, disease, infection, or other trauma often requires treatment to cover the affected area. For example, physicians use skin grafts obtained from the patient's own skin or from external sources. However, skin grafts obtained from the patient's own skin typically result in large scars in the area from which the skin was taken, e.g., the thigh, back, or buttocks. Skin grafts can also be taken from donors or cadavers, but there are issues such as the donor's health, absence of infectious diseases, and extensive screening to determine the compatibility of the donor skin graft with the patient's skin, as well as the possibility of immunological rejection by the patient of the donor skin graft.

[0002] Additionally, current skin grafts require surgery under general or local anesthesia, which can lead to complications such as severe pain, infection, bleeding, additional surgery, and, in rare cases, death, as well as additional hardships such as hospitalization, healing periods, and scarring at the donor site.

[0003] Thus, there remains a need for new skin graft compositions. Summary of the Invention

[0004] The present invention provides a method of preparing a skin graft composition, comprising: (a) obtaining a skin sample from a subject in need of a skin layer patch or dressing; (b) placing the skin sample in a container with a sterile medium; (c) isolating fibroblasts and keratinocytes from the skin sample to obtain a solution containing fibroblasts and keratinocytes or reshaping and resizing the skin sample to obtain a solution containing a reshaped and resized skin sample; and (d) combining the solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample with a matrix to form a skin graft composition. The present invention also provides a method of providing a skin graft composition to a subject, and a method of providing a skin layer patch or dressing, or a skin graft to a subject in need of a skin graft.

[0005] Detailed Description of the Invention The skin graft composition of the present invention is prepared by a method comprising, consisting essentially of, or consisting of: (a) obtaining a skin sample from a subject in need of a skin layer patch or dressing; (b) placing the skin sample in a container with a sterile medium; (c) isolating fibroblasts and keratinocytes from the skin sample to obtain a solution comprising fibroblasts and keratinocytes or reshaping and resizing the skin sample to obtain a solution comprising a reshaped and resized skin sample; and (d) combining the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample with a matrix to form the skin graft composition.

[0006] The method includes obtaining a skin sample from a subject in need of a skin layer patch or dressing. The subject can be any mammal, preferably a human, in need of a skin layer patch or dressing. The skin layer patch or dressing can be a skin graft. The skin sample can be any suitable sample. The skin sample can be obtained from any suitable location of the patient. In some embodiments, the skin sample is obtained from the head, back, buttocks, or legs, such as the thighs. In some embodiments, the skin sample includes the epidermis, dermis, and subcutaneous tissue. In other embodiments, the skin sample includes the epidermis and dermis. In other embodiments, the skin sample includes the epidermis and at least a portion of the dermis, but does not include the entire dermis.

[0007] Skin samples can be obtained by any suitable means using any suitable tool. For example, in some embodiments, skin samples are obtained using a razor or similar tool, a skin punch (also called a biopsy punch or surgical punch), or a scalpel. In other embodiments, skin samples are obtained using a dermatome, such as a Goulian knife or an air dermatome.

[0008] The skin sample can be of any suitable size. In some embodiments, the skin sample is at least 1 mm 2 For example, in some embodiments, the skin sample has a surface area of ​​at least 1 mm 2 , at least 2 mm 2 , at least 3 mm 2 , at least 4 mm 2 , at least 5 mm 2 , at least 6mm 2 , at least 7mm 2 , at least 8mm 2 , at least 9mm 2 , at least 10 mm 2 , at least 11 mm 2 , at least 12 mm 2 , at least 13mm 2 , at least 14mm 2, at least 15 mm 2 , at least 16mm 2 , at least 17mm 2 , at least 18mm 2 , at least 19mm 2 , at least 20 mm 2 , at least 100 mm 2 , at least 500 mm 2 , at least 1000mm 2 , at least 1500mm 2 , at least 2000mm 2 , at least 2500mm 2 , at least 3000mm 2 , at least 4000mm 2 , at least 5000mm 2 , or at least 10000mm 2 or a surface area between any two of the aforementioned maximums. In some embodiments, the sample has a length between 1 mm and 10 mm, e.g., 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm, or a length within a range defined by any two of the aforementioned values. In some embodiments, the sample has a width between 1 and 10 mm, e.g., 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm, or a width within a range defined by any two of the aforementioned values. In some embodiments, the skin sample has a thickness between 2 and 3 mm. For example, in some embodiments, the sample has a thickness of 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, or 3 mm, or a thickness within a range defined by any two of the aforementioned values. In some embodiments, the skin sample has a thickness of 1 mm or less. For example, in some embodiments, the sample has a thickness of 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm, or a thickness within a range defined by any two of the foregoing values.

[0009] In some embodiments, the method includes dissecting the skin sample prior to placing the skin sample in a container with a sterile medium. The skin sample can be dissected by any suitable means. In some embodiments, the skim sample is dissected using a scalpel or knife.

[0010] The method includes placing the skin sample in a container with a sterile medium. The container can be any suitable container. In some embodiments, the container is a glass or plastic container. In some embodiments, the container is a cryovial or test tube. In other embodiments, the container is a cell culture flask or roller bottle. In some embodiments, the container is a petri dish or cell culture plate. In other embodiments, the container is a cell culture insert placed in a cell culture plate.

[0011] The sterile medium can be any suitable sterile medium. A sterile medium is a medium that does not contain viable bacteria or other cells that grow and increase in number by cell division. In some embodiments, the sterile medium is saline. In some embodiments, the saline is phosphate buffered saline (PBS). In other embodiments, the sterile medium is Dulbecco's Modified Eagle's Medium (DMEM). In other embodiments, the sterile medium is Ham's F-12 (F12), Ham's F-10 (F10), Iscove's Modification of DMEM (IDMEM), Medium 199, Alpha MEM, Basal Medium Eagle with Earle's BSS: Cryoprotective Medium (Freezing Medium), DMEM:F12 1:1 with L-glutamine, Glasgow's MEM with L-glutamine (GMEM), IMDM with HEPES (with or without L-glutamine):L-15 (Leibovitz) without L-glutamine, McCoy's 5A Modification, Medium 199, MEM Eagle, MEM Eagle-Earle's BSS with or without L-glutamine, MEM Eagle-Hank's BSS, NCTC-109, Richter's CM Medium, RPMI 1640 with HEPES, RPMI 1640. In some embodiments, the sterile medium is a combination of sterile media, such as a combination of one or more of the aforementioned sterile media. The amount of sterile medium can be any suitable amount of sterile medium. In some embodiments, the volume of sterile medium is between 1 and 10,000 μl / mm 2 A skin sample, e.g., 1 μl / mm 2 Skin sample, 500 μl / mm 2 Skin sample, 1000 μl / mm 2 Skin sample, 1500 μl / mm 2 Skin sample, 2000 μl / mm 2 Skin sample, 2500 μl / mm 2 Skin sample, 3000 μl / mm 2 Skin sample, 3500 μl / mm 2 Skin sample, 4000 μl / mm 2 Skin sample, 4500 μl / mm 2 Skin sample, 5000 μl / mm 2 Skin sample, 5500 μl / mm 2 Skin sample, 6000 μl / mm2 Skin sample, 6500 μl / mm 2 Skin sample, 7000 μl / mm 2 Skin sample, 7500 μl / mm 2 Skin sample, 8000 μl / mm 2 Skin sample, 8500 μl / mm 2 Skin sample, 9000 μl / mm 2 Skin sample, 9500 μl / mm 2 Skin sample, or 10,000 μl / mm 2 the skin sample, or a volume of sterile medium within a range defined by any two of the preceding values.

[0012] The skin sample can be maintained in the container with the sterile medium for any suitable period of time before isolating fibroblasts and keratinocytes from the skin sample or before reshaping and resizing the skin sample. In some embodiments, the skin sample is maintained in the container for up to 72 hours. For example, in some embodiments, the skin sample is maintained in the container for 0 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours, or a time within a range defined by any two of the aforementioned values. The skin sample can be maintained in the container with the sterile medium at any suitable temperature. In some embodiments, the skin sample is maintained in the container at a temperature of 5-40°C, for example 5-37°C. In some embodiments, the skin sample is maintained in the container at a temperature of 20-40°C. For example, in some embodiments, the skin sample is maintained in the container at a temperature of 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., 36° C., 37° C., 38° C., 39° C., or 40° C., or a temperature within a range defined by any two of the foregoing values. In a preferred embodiment, the skin sample is maintained in the container at a temperature of 37° C.

[0013] In some embodiments, the method further comprises adding additional sterile medium to the container prior to isolating fibroblasts and keratinocytes from the skin sample or prior to reshaping and resizing the skin sample. The additional sterile medium can be any suitable sterile medium. The additional sterile medium can be the same or different from the initial amount of sterile medium added to the container. A sterile medium is a medium that does not contain live bacteria or other cells that grow and increase in number by cell division. In some embodiments, the sterile medium is saline. In some embodiments, the saline is phosphate buffered saline (PBS). In other embodiments, the sterile medium is Dulbecco's modified Eagle's medium (DMEM). In other embodiments, the sterile medium is Ham's F-12 (F12), Ham's F-10 (F10), Iscove's Modification of DMEM (IDMEM), Medium 199, AlphaMEM, Basal Medium Eagle with Earle's BSS: Cryoprotective Medium (Freezing Medium), DMEM:F12 1:1 with L-glutamine, Glasgow's MEM with L-glutamine (GMEM), IMDM with HEPES (with or without L-glutamine):L-15 (Leibovitz) without L-glutamine, McCoy's 5A Modification, Medium 199, MEM Eagle, MEM Eagle-Earle's BSS with or without L-glutamine, MEM Eagle-Hank's BSS, NCTC-109, Richter's CM Medium, RPMI 1640 with HEPES, RPMI 1640. In some embodiments, the sterile medium is a combination of sterile media, such as a combination of one or more of the aforementioned sterile media. The amount of additional sterile medium can be any suitable amount. In some embodiments, the amount of additional sterile medium is between 1 and 10,000 μl / mm 2 A skin sample, e.g., 1 μl / mm 2 Skin sample, 500 μl / mm 2 Skin sample, 1000 μl / mm 2 Skin sample, 1500 μl / mm 2 Skin sample, 2000 μl / mm 2 Skin sample, 2500 μl / mm 2 Skin sample, 3000 μl / mm 2 Skin sample, 3500 μl / mm2 Skin sample, 4000 μl / mm 2 Skin sample, 4500 μl / mm 2 Skin sample, 5000 μl / mm 2 Skin sample, 5500 μl / mm 2 Skin sample, 6000 μl / mm 2 Skin sample, 6500 μl / mm 2 Skin sample, 7000 μl / mm 2 Skin sample, 7500 μl / mm 2 Skin sample, 8000 μl / mm 2 Skin sample, 8500 μl / mm 2 Skin sample, 9000 μl / mm 2 Skin sample, 9500 μl / mm 2 Skin sample, or 10,000 μl / mm 2 the skin sample, or a volume of sterile medium within a range defined by any two of the preceding values.

[0014] The method includes isolating fibroblasts and keratinocytes from a skin sample to obtain a solution containing fibroblasts and keratinocytes, or reshaping and resizing a skin sample to obtain a solution containing a reshaped and resized skin sample. In some embodiments, the method includes isolating fibroblasts and keratinocytes from a skin sample to obtain a solution containing fibroblasts and keratinocytes. The fibroblasts and keratinocytes can be separated from the skin sample by any suitable method. In some embodiments, the fibroblasts and keratinocytes are separated from the skin sample by mechanical, chemical, or enzymatic extraction. In some embodiments, the fibroblasts and keratinocytes are separated from the skin sample by mechanical extraction. In some embodiments, the fibroblasts and keratinocytes are separated from the skin by enzymatic extraction. In some embodiments, only some types of extraction are used. For example, in some embodiments, mechanical extraction, chemical extraction, or enzymatic extraction are used. In other embodiments, more than one type of extraction is used. For example, in some embodiments, mechanical and chemical extraction, mechanical and enzymatic extraction, or chemical and enzymatic extraction are used. In other embodiments, three or more types of extraction are used. For example, in some embodiments, mechanical, chemical, and enzymatic extraction are used.

[0015] In some embodiments, mechanical extraction is used to separate or dissociate fibroblasts and keratinocytes from skin samples. Any suitable method of mechanical extraction can be used. For example, in some embodiments, the mechanical extraction is shaking, stirring, mixing, grinding, or mechanical pipetting.

[0016] In some embodiments, the container containing the skin sample and the sterile medium is shaken. The container containing the skin sample and the sterile medium is shaken for any suitable time to separate the fibroblasts and keratinocytes from the remainder of the skin sample. In some embodiments, the container containing the skin sample and the sterile medium is shaken for up to 1800 seconds (s). In some embodiments, the container containing the skin sample and the sterile medium is shaken for up to one of the following periods: 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 1100s, 1200s, 1300s, 1400s, 1500s, 1600s, 1700s, or 1800s, or for a time defined by any two of the aforementioned values. For example, in some embodiments, the container containing the skin sample and the sterile medium is shaken for between 5 and 100s. In some embodiments, the container containing the skin sample and the sterile medium is shaken for up to one of the following periods: 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, 70 s, or 80 s, or for a time defined by any two of the aforementioned values. For example, in some example embodiments, the container containing the skin sample and the sterile medium is shaken for 30-40 s, e.g., 37 s. In other embodiments, the container containing the skin sample and the sterile medium is shaken for 60-70 s, e.g., 64 s. The container containing the skin sample and the sterile medium may be shaken by any suitable means by any suitable device. In some embodiments, the container is shaken manually. In some embodiments, the container is shaken using a device such as a laboratory shaker or dissociator.

[0017] In other embodiments, the skin sample and sterile medium in the container are agitated or mixed. The skin sample and sterile medium in the container are agitated or mixed for any suitable time to separate the fibroblasts and keratinocytes from the remainder of the skin sample. In some embodiments, the skin sample and sterile medium in the container are agitated or mixed for up to 1800 seconds (s). In some embodiments, the skin sample and sterile medium in the container are agitated or mixed for up to one of the following periods: 100 s, 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, 1000 s, 1100 s, 1200 s, 1300 s, 1400 s, 1500 s, 1600 s, 1700 s, or 1800 s, or for a time defined by any two of the aforementioned values. For example, in some embodiments, the skin sample and sterile medium in the container are agitated or mixed for between 5 and 100 s. In some embodiments, the skin sample and sterile medium in the container are stirred or mixed for up to one of the following periods: 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, 70 s, or 80 s, or for a time defined by any two of the aforementioned values. For example, in some embodiments, the skin sample and sterile medium in the container are stirred or mixed for 30-40 s, e.g., 37 s. In other embodiments, the skin sample and sterile medium in the container are stirred or mixed for 60-70 s, e.g., 64 s. The skin sample and sterile medium in the container are stirred or mixed by any suitable means by any suitable device. In some embodiments, the skin sample and sterile medium are stirred or mixed manually. In some embodiments, the skin sample and sterile medium in the container are stirred or mixed using a laboratory stirrer, mixer, dissociator, or the like, or a stirring bar.

[0018] In other embodiments, the mechanical extraction is mechanical pipetting.In other embodiments, the mechanical extraction is grinding the sample using any suitable grinder or mill.

[0019] In some embodiments, chemical extraction is used to separate or dissociate fibroblasts and keratinocytes from the skin sample. Any suitable method of chemical extraction can be used. In some embodiments, the chemical extraction involves incubating the skin sample and sterile medium in a container with a chemical at a suitable temperature for a suitable period of time. In some embodiments, the chemical is ethylenediaminetetraacetic acid (EDTA) and / or egutamic acid (EGTA). In some embodiments, the chemical extraction is performed for a time ranging from 1 minute to 24 hours. In some embodiments, the chemical extraction is performed at a temperature of 5 to 50° C., for example, 5 to 37° C. In some embodiments, the chemical extraction is carried out at a temperature between 20 and 40° C., e.g., 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., 36° C., 37° C., 38° C., 39° C., or 40° C., or a temperature within a range defined by any two of the preceding values. In a preferred embodiment, the chemical extraction is carried out at a temperature of 37° C.

[0020] In some embodiments, enzymatic extraction is used to separate or dissociate fibroblasts and keratinocytes from the sample. Any suitable method of enzymatic extraction can be used. In some embodiments, the skin sample and sterile medium in a container are incubated with an enzyme at a suitable temperature for a suitable period of time. In some embodiments, the enzyme is trypsin, chymotrypsin, papain, chymopapain, collagenase, elastase, hyaluronidase, D-nase, pronase, and / or dispase. In some embodiments, the enzymatic extraction is performed for a period of time ranging from 1 minute to 24 hours. In some embodiments, the enzymatic extraction is performed at a temperature of 5 to 50°C, for example, 5 to 37°C. In some embodiments, the enzymatic extraction is carried out at a temperature between 20 and 40° C., e.g., 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., 36° C., 37° C., 38° C., 39° C., or 40° C., or a temperature within a range defined by any two of the preceding values. In a preferred embodiment, the enzymatic extraction is carried out at a temperature of 37° C.

[0021] The solution comprising fibroblasts and keratinocytes formed after isolating the fibroblasts and keratinocytes from the skin sample is free or at least substantially free of microorganisms. In some embodiments, the solution comprising fibroblasts and keratinocytes formed after isolating the fibroblasts and keratinocytes from the skin sample is free or at least substantially free of bacteria. In some embodiments, the solution comprising fibroblasts and keratinocytes formed after isolating the fibroblasts and keratinocytes from the skin sample is free or at least substantially free of viruses.

[0022] In some embodiments, the solution containing fibroblasts and keratinocytes is substantially free or free of other skin cells. In some embodiments, the solution formed after separation of fibroblasts and keratinocytes from the skin sample contains bacteria and optionally other microorganisms. In such embodiments, the method for preparing a skin graft composition further comprises removing bacteria and optionally other microorganisms from the solution formed after separation of fibroblasts and keratinocytes from the skin sample, such that the solution is free or at least substantially free of bacteria and optionally other microorganisms. Any suitable method can be used to remove bacteria and optionally other microorganisms. In some embodiments, the bacteria and optionally other microorganisms are removed using any suitable antibacterial or antimicrobial agent. In some embodiments, the antibacterial or antimicrobial agent is penicillin, streptomycin, gentamicin sulfate, tobramycin, and / or amphotericin B. As used herein, substantially free of bacteria or optionally other microorganisms refers to less than 100 bacteria or other microorganisms per ml. For example, less than one of 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 microbial loads, or a bacterial count or any other microbial count within a range defined by any two of the foregoing values.

[0023] In some embodiments, the fibroblast- and keratinocyte-containing solution formed after separating the fibroblasts and keratinocytes from the skin sample also contains the remainder of the skin sample, such as hair follicles, adipocytes, melanocytes, capillaries and non-fibroblastic connective tissue cells.

[0024] In some embodiments, the method includes reshaping and resizing the skin sample and obtaining a solution containing the reshaped and resized skin sample. The skin sample may be reshaped and resized in any suitable manner. In some embodiments, the skin sample is reshaped and resized by cutting the skin sample into smaller pieces. The skin sample may be cut using any suitable tool, such as a scalpel. In other embodiments, the skin sample is reshaped and resized using a dissociator. For example, in some embodiments, a container with the skin sample and sterile medium is placed in the dissociator. In some embodiments, the container with the skin sample and sterile medium is placed in the dissociator for any suitable time. In some embodiments, the skin sample is placed in the dissociator for any time between 10 seconds and 2 minutes, such as 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, or 120 seconds, or within a range defined by any two of the aforementioned values. In some embodiments, the skin sample is placed in the dissociator for 50-60 seconds, e.g., 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 56 seconds, 57 seconds, 58 seconds, 59 seconds, or 60 seconds, or a period within a range defined by any two of the foregoing values. In some embodiments, the reshaped and resized skin sample maintains all layers of the skin sample.

[0025] In some embodiments, the skin sample is removed from the container and sterile medium prior to reshaping and resizing the skin sample, and replaced with the container and sterile medium after reshaping and resizing the skin sample, In some embodiments, the skin sample is replaced into an empty container, and additional sterile medium is added to the container as described herein to form a solution containing the reshaped and resized skin sample.

[0026] In some embodiments, the method of preparing a skin graft composition further comprises filtering and / or centrifuging the solution comprising fibroblasts and keratinocytes or the solution comprising the remodeled and resized skin sample to provide a purified solution of fibroblasts and keratinocytes or the purified solution comprising the remodeled and resized skin sample. In some embodiments, the purified solution of fibroblasts and keratinocytes is a solution comprising fibroblasts and keratinocytes that is free or at least substantially free of one or more of melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells. Desirably, the purified solution of fibroblasts and keratinocytes is a solution comprising fibroblasts and keratinocytes that is free or at least substantially free of melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells. In some embodiments, a solution that is substantially free of melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells is a solution that has fewer than 10000 melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells. In some embodiments, a solution that is substantially free of melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells is a solution that has fewer than 9000, fewer than 8000, fewer than 7000, fewer than 6000, fewer than 5000, fewer than 4000, fewer than 3000, fewer than 2000, or fewer than 1000 melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells, or a solution in which the melanocytes, adipocytes, hair follicles, capillaries, and non-fibroblastic connective tissue cells are within a range defined by any two of the foregoing values.

[0027] In some embodiments, the method of preparing a skin graft composition further comprises filtering the solution comprising fibroblasts and keratinocytes to provide a purified solution of fibroblasts and keratinocytes, or filtering the solution comprising the reshaped and resized skin sample to provide a purified solution of the reshaped and resized skin sample. Any suitable filter may be used to provide the purified solution of fibroblasts and keratinocytes. In some embodiments, the filter has a pore size of 1-200 microns. In some embodiments, the filter has a pore size of 10-100 microns, e.g., 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, or 100 microns, or a pore size within a range defined by any two of the aforementioned values.

[0028] In some embodiments, the method of preparing a skin graft composition further comprises centrifuging the solution comprising fibroblasts and keratinocytes to provide a purified solution of fibroblasts and keratinocytes, or centrifuging the solution comprising the reshaped and resized skin sample to provide a purified solution comprising the reshaped and resized skin sample. The solution comprising fibroblasts and keratinocytes, or the solution comprising the reshaped and resized skin sample, can be centrifuged for any suitable time and with any suitable force. In some embodiments, the solution comprising fibroblasts and keratinocytes, or the solution comprising the reshaped and resized skin sample, is centrifuged for 1-60 minutes. In some embodiments, the solution comprising fibroblasts and keratinocytes, or the solution comprising the reshaped and resized skin sample, is centrifuged for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes, or for a time within a range defined by any two of the aforementioned values. In some embodiments, the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample is centrifuged at 1-400 g. In some embodiments, the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample is centrifuged at 1-300 g. In some embodiments, the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample is centrifuged at 100-300 g. In some embodiments, the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample is centrifuged at 150-300 g. In some embodiments, the solution containing fibroblasts and keratinocytes, or the solution containing the reshaped and resized skin sample, is centrifuged at a force between 180 and 300 g, e.g., 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, or 300 g, or within a range defined by any two of the foregoing values.

[0029] In some embodiments, the method includes centrifuging the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample at least one additional time, for example at least one additional time, at least two additional times, at least three additional times, or at least four additional times. The purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample can be centrifuged for any suitable period of time and at any suitable force. For example, in some embodiments, the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample is centrifuged at least one additional time for 1 to 60 minutes. In some embodiments, the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample is centrifuged at least one additional time for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes, or within a range defined by any two of the aforementioned values. In some embodiments, the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample is centrifuged at least one additional time between 1 and 400 g. In some embodiments, the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample is centrifuged at 1 to 300 g. In some embodiments, the solution comprising fibroblasts and keratinocytes or the solution comprising the reshaped and resized skin sample is centrifuged at 100 to 300 g. In some embodiments, the purified solution comprising fibroblasts and keratinocytes or the purified solution comprising the reshaped and resized skin sample is centrifuged at least one additional time between 150 to 300 g.In some embodiments, the purified solution containing fibroblasts and keratinocytes, or the purified solution containing the reshaped and resized skin sample, is centrifuged at least one additional time at a force between 180 and 300 g, e.g., 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, or 300 g, or within a range defined by any two of the foregoing values.

[0030] In some embodiments, the method includes filtering the solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample to provide a purified solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample, and then centrifuging the purified solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample. In other embodiments, the method includes filtering the solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample to provide a purified solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample, and then centrifuging the purified solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample at least twice. In some embodiments, the method includes removing visible skin debris from the purified solution containing fibroblasts and keratinocytes or the solution containing the reshaped and resized skin sample after the first centrifugation and before the second centrifugation.

[0031] In some embodiments, the separation of fibroblasts and keratinocytes from the skin sample results in the fibroblasts and keratinocytes being viable, alive, and / or intact. In some embodiments, the separation of fibroblasts and keratinocytes from the skin sample results in the fibroblasts and keratinocytes being viable. In some embodiments, the separation of fibroblasts and keratinocytes results in the fibroblasts and keratinocytes being viable. In some embodiments, the separation of fibroblasts and keratinocytes results in the fibroblasts and keratinocytes being intact. In some embodiments, the use of the skin graft composition or skin graft described herein in which the fibroblasts and keratinocytes are viable, alive, and / or intact results in epithelialization, otherwise known as the process by which epithelial cells migrate to the top of an open wound to close it. In some embodiments, use of the skin graft compositions or skin grafts described herein in which fibroblasts and keratinocytes are viable, alive, and / or intact results in little to no scar tissue formation.

[0032] In some other embodiments, separation of fibroblasts and keratinocytes from the skin sample results in lysis, death, and / or decomposition of all, or at least substantially all, cells of the skin sample, including fibroblasts and keratinocytes.

[0033] In some embodiments, the cells in the solution containing the reshaped and resized skin sample are lysed after centrifugation. The cells in the reshaped and resized skin sample are lysed by any suitable means.

[0034] In some embodiments, the use of a skin graft composition or skin graft described herein in which all or substantially all of the cells in a skin sample have been lysed results in wound closure and scar tissue formation at the application site. Without being bound to a particular theory, it is believed that the lysed cells release fibroblast recruitment proteins into solution, which when applied to a subject as part of a skin graft composition described herein, aid in the formation of scar tissue at the application site. In some embodiments, scar tissue formation occurs at the site of an open wound. In some embodiments, the open wound is a wound that has been open for at least one week. For example, in some embodiments, the wound is open for at least one of the following values, or for a period within a range defined by any two of the aforementioned values: 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, or 36 weeks. In other embodiments, the wound is an open wound for at least 3 months, e.g., 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 5 years, 10 years, 20 years, 30 years, or 40 years, or for a period within a range defined by any two of the foregoing values. In some embodiments, the open wound is a chronic wound.

[0035] The method includes combining a solution including fibroblasts and keratinocytes or a solution including a reshaped and resized skin sample with a matrix to form a skin graft composition. In some embodiments, the solution including fibroblasts and keratinocytes is a purified solution including fibroblasts and keratinocytes as described herein, which is combined with a matrix to form a skin graft composition. In some embodiments, the solution including a reshaped and resized skin sample is a purified solution including a reshaped and resized skin sample as described herein, which is combined with a matrix to form a skin graft composition. In other embodiments, the method further includes isolating or substantially purifying the fibroblasts and keratinocytes from the solution including fibroblasts and keratinocytes, and then combining the isolated or substantially purified fibroblasts and keratinocytes with a matrix. In other embodiments, the method further includes isolating or substantially purifying the reshaped and resized skin sample from the solution including a reshaped and resized skin sample, and then combining the isolated or substantially purified skin sample with a matrix.

[0036] The matrix can be any suitable matrix. In some embodiments, the matrix is ​​a fluid or gel, such as a fluid or gel matrix that conforms to the surface to which the skin graft composition is applied. For example, in some embodiments, the matrix conforms to the wound. In some embodiments, the matrix is ​​a gel matrix. In some embodiments, the matrix is ​​a colloidal solution. In some embodiments, the matrix comprises, consists essentially of, or consists of collagen, gelatin, agarose, cellulose, elastin, fibrin, proteoglycans such as decorin or biglycan, glycoproteins such as tenascin, vitronectin, fibronectin, laminin, or thrombospondin I, glycosaminoglycans (GAGs) such as hyaluronic acid (HA), polyacids such as polylactic acid, polyglycolic acid, or polyamino acids, polycaprolactone, polyamino acids, polypeptide gels, synthetic polymeric fibers made from copolymers thereof, alone or in combination. In some embodiments, the matrix is ​​a collagen solution. In some embodiments, the collagen solution comprises type I collagen, type II collagen, type III collagen, or type IV collagen. In some embodiments, the collagen solution comprises type I collagen. In some embodiments, the matrix is ​​a fibrin solution. In some embodiments, the matrix is ​​a keratin solution. In some embodiments, the matrix is ​​a solution comprising, consisting essentially of, or consisting of collagen, fibrin, and keratin. In some embodiments, the matrix is ​​a solution comprising, consisting essentially of, or consisting of collagen and fibrin. In some embodiments, the matrix is ​​a solution comprising, consisting essentially of, or consisting of collagen and keratin. In some embodiments, the matrix is ​​a solution comprising, consisting essentially of, or consisting of fibrin and keratin. In some embodiments, the matrix does not comprise any components other than those mentioned.

[0037] The matrix can have any suitable pH. In some embodiments, the matrix has a pH of 6-8. In some embodiments, the matrix has a pH of 6.8-7.4, such as pH 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4, or a pH within a range defined by any two of the foregoing values. The matrix can have any suitable viscosity. In some embodiments, the matrix has a viscosity of 0.5-1,000,000 cP. In some embodiments, the matrix has a viscosity of 2,000-250,000 cP. In some embodiments, the matrix has a viscosity of 10,000 to 100,000 cP, e.g., 10,000 cP, 15,000 cP, 20,000 cP, 25,000 cP, 30,000 cP, 35,000 cP, 40,000 cP, 45,000 cP, 50,000 cP, 55,000 cP, 60,000 cP, 65,000 cP, 70,000 cP, 7500 cP, 80,000 cP, 85,000 cP, 90,000 cP, 95,000 cP, or 100,000 cP, or a viscosity within a range defined by any two of the foregoing values.

[0038] After the solution or purified solution containing fibroblasts and keratinocytes or the solution or purified solution containing remodeled and resized skin sample is combined with the matrix, in some embodiments, the concentration of the matrix in the matrix containing fibroblasts and keratinocytes or the matrix containing skin sample is 100mg / ml or less. In some embodiments, the concentration of the matrix is ​​90mg / ml, 80mg / ml, 70mg / ml, 60mg / ml, 50mg / ml, 40mg / ml, 30mg / ml, 20mg / ml, or 10mg / ml or less, or within a range defined by any two of the aforementioned values. In a preferred embodiment, the concentration of the matrix is ​​65mg / ml. In some embodiments, the concentration of the matrix containing fibroblasts and keratinocytes or the matrix containing remodeled and resized skin sample is 10mg / ml or less. For example, in some embodiments, the concentration of the matrix is ​​less than or equal to one of the following values: 10 mg / ml, 9 mg / ml, 8 mg / ml, 7 mg / ml, 6 mg / ml, 5 mg / ml, 4 mg / ml, 3 mg / ml, 2 mg / ml, or 1 mg / ml, or a concentration within a range defined by any two of the foregoing values.

[0039] In some embodiments, the matrix comprising the fibroblasts and keratinocytes, or the matrix comprising the remodeled and resized skin sample, is a fluid or gel. In some embodiments, the matrix comprising the fibroblasts and keratinocytes, or the matrix comprising the remodeled and resized skin sample, is a fluid or gel that desirably conforms to the surface to which the skin graft composition is applied, such as a wound.

[0040] In some embodiments, the method further comprises culturing the matrix comprising fibroblasts and keratinocytes, or the matrix comprising the reshaped and resized skin sample. The matrix comprising fibroblasts and keratinocytes, or the matrix comprising the reshaped and resized skin sample, can be cultured for any suitable time and under any suitable conditions. In some embodiments, the matrix comprising fibroblasts and keratinocytes, or the matrix comprising the reshaped and resized skin sample, is cultured for a period of up to 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours, or for a time within a range defined by any two of the aforementioned values. In some embodiments, the matrix comprising fibroblasts and keratinocytes, or the matrix comprising the reshaped and resized skin sample, is cultured for 24 to 28 hours. In some embodiments, the matrix containing fibroblasts and keratinocytes, or the matrix containing the reshaped and resized skin sample, is cultured at a temperature between 5 and 40° C., for example, between 5 and 37° C. In some embodiments, the matrix containing fibroblasts and keratinocytes, or the matrix containing the reshaped and resized skin sample, is cultured at a temperature between 20 and 40° C. For example, in some embodiments, the matrix containing fibroblasts and keratinocytes, or the matrix containing the reshaped and resized skin sample, is cultured at a temperature of 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., 36° C., 37° C., 38° C., 39° C., or 40° C., or a temperature within a range defined by any two of the aforementioned values. In a preferred embodiment, the matrix containing fibroblasts and keratinocytes, or the matrix containing the remodeled and resized skin sample, is cultured at a temperature of 37°C.In some embodiments, the matrix containing fibroblasts and keratinocytes or the matrix containing the remodeled and resized skin sample is cultured in an environment of 0-20% CO2. In some embodiments, the matrix containing fibroblasts and keratinocytes or the matrix containing the remodeled and resized skin sample is cultured in an environment of 0-10% CO2, such as 0% CO2, 1% CO2, 2% CO2, 3% CO2, 4% CO2, 5% CO2, 6% CO2, 7% CO2, 8% CO2, 9% CO2, or 10% CO2, or an environment with a CO2 concentration within a range defined by any two of the aforementioned values. In a preferred embodiment, the matrix containing fibroblasts and keratinocytes or the matrix containing the remodeled and resized skin sample is cultured in an environment of 5% CO2.

[0041] In other embodiments, the method does not include culturing a matrix comprising fibroblasts and keratinocytes or a matrix comprising a reshaped and resized skin sample. In some embodiments, the fibroblasts and keratinocytes are only modified as described herein. In other embodiments, the reshaped and resized skin sample is only modified as described herein. For example, in some embodiments, the fibroblasts and keratinocytes are only separated from the skin sample to obtain a solution comprising fibroblasts and keratinocytes, and the solution comprising fibroblasts and keratinocytes is combined with the matrix to thereby form the skin graft composition. In other embodiments, the skin sample is only reshaped and resized to obtain a solution comprising a reshaped and resized skin sample, and the solution comprising the reshaped and resized skin sample is combined with the matrix to form the skin graft composition.

[0042] The present invention provides a skin graft composition. In one embodiment, the skin graft composition is prepared by any of the methods described herein. In another embodiment, the skin graft composition comprises a matrix and a skin sample in which all, or at least substantially all, of the cells of the skin sample have been lysed. The lysed cells of the skin sample can be uniformly dispersed in the matrix.

[0043] In yet another embodiment, a skin graft composition comprises (a) a matrix and (b) fibroblasts and keratinocytes isolated from a skin sample, wherein the composition contains less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml. For example, in some embodiments, the skin graft composition contains less than 10,000, less than 9,000, less than 8,000, less than 7,000, less than 6,000, less than 5,000, less than 4,000, less than 3,000, less than 2,000, or less than 1,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml, or the amount of hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml is within a range defined by any two of the aforementioned values.

[0044] In yet another embodiment, a skin graft composition comprises (a) a matrix and (b) a reshaped and resized skin sample, wherein the composition contains less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml. For example, in some embodiments, the skin graft composition contains less than 10,000, less than 9,000, less than 8,000, less than 7,000, less than 6,000, less than 5,000, less than 4,000, less than 3,000, less than 2,000, or less than 1,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml, or the amount of hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblastic connective tissue cells per ml is within a range defined by any two of the aforementioned values.

[0045] In any embodiment, the skin graft composition may be free or substantially free of bacteria or optionally other microorganisms. Substantially free of bacteria or optionally other microorganisms refers to less than 100 bacteria or optionally other microorganisms per ml, such as less than one of the following amounts: 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 bacteria or optionally other microorganisms, or a number of bacteria or optionally other microorganisms within a range defined by any two of the preceding values.

[0046] In either embodiment, the fibroblasts and keratinocytes can be uniformly dispersed throughout the matrix.

[0047] The skin graft compositions provided by the present invention can be used in the preparation of a skin graft onto a subject.

[0048] The present invention provides a method of providing a skin layer patch or dressing, which may be a skin graft, to a subject, comprising providing a skin graft composition according to the methods as described herein and applying the skin graft composition to a surface of a subject in need of the skin layer patch or dressing.

[0049] The present invention provides a method for preparing a skin layer patch or dressing, comprising providing a skin graft composition as described herein and applying the skin graft composition to any suitable surface. In some embodiments, the surface of the subject is a surface that requires coverage and protection that would typically be provided by healthy skin. For example, the surface of the subject may be a surface that requires a skin layer patch or dressing, such as a surface that requires a skin graft. In some embodiments, the surface is an open wound. In some embodiments, the surface is a chronic wound. In other embodiments, the surface is a surface that requires epithelialization, such as a wrinkled surface.

[0050] In embodiments of the invention, the skin graft composition is applied to a surface of a subject requiring a skin layer patch or dressing, such as a surface of a subject requiring a skin graft, by any suitable method and any suitable means. For example, the skin graft composition can be extruded from a press tube onto the surface of the subject. Any suitable press tube can be used. In some embodiments, the press tube is a syringe. In some embodiments, the press tube is any cartridge having a plunger. In some embodiments, the press tube is a pouch. In other embodiments, the skin graft composition is applied by spreading the skin graft composition onto the surface of the subject with any suitable tool, such as a spatula. In other embodiments, the skin graft composition is applied by pouring the skin graft composition onto the surface of the subject.

[0051] Embodiment

[0052] 1. A method of preparing a skin graft composition comprising: (a) obtaining a skin sample from a subject requiring a skin layer patch or dressing; (b) placing the skin sample in a container containing a sterile medium; (c) isolating fibroblasts and keratinocytes from the skin sample to obtain a solution containing fibroblasts and keratinocytes or reshaping and resizing the skin sample to obtain a solution containing a reshaped and resized skin sample; and (d) combining the solution containing fibroblasts and keratinocytes, or the solution containing the remodeled and resized skin sample, with a matrix to form a skin graft composition.

[0053] 2. The method of embodiment 1, wherein the fibroblasts and keratinocytes from the skin sample are separated to obtain a solution containing the fibroblasts and keratinocytes, and the solution containing the fibroblasts and keratinocytes is combined with a matrix, thereby forming a skin graft composition.

[0054] 3. The method of embodiment 2, wherein after isolating the fibroblasts and keratinocytes from the skin sample, the fibroblasts and keratinocytes are viable.

[0055] 4. The method of embodiment 2 or 3, wherein the skin sample is dissected prior to placing the skin sample in the container with the sterile medium.

[0056] 5. Skin samples must be at least 1 mm 2 The method of any one of embodiments 2 to 4, wherein

[0057] 6. The method of any of embodiments 2-5, wherein the container is a glass container or a plastic container.

[0058] 7. The method of any of embodiments 2-5, wherein the container is a cryovial or a test tube.

[0059] 8. The method of any of embodiments 2-7, wherein the sterile medium is saline.

[0060] 9. The method of embodiment 8, wherein the saline is phosphate buffered saline (PBS).

[0061] 10. The method of any of embodiments 2-7, wherein the sterile medium is Dulbecco's Modified Eagle's Medium (DMEM).

[0062] 11. The amount of sterile medium is enough to cover 1 mm of skin sample. 2 The method of any one of embodiments 2 to 10, wherein the amount of the solution is 1 to 10,000 μl per unit volume.

[0063] 12. The method of any of embodiments 2-11, wherein the skin sample is maintained in a container with a sterile medium for up to 72 hours prior to isolating fibroblasts and keratinocytes from the skin sample.

[0064] 13. The method of any of embodiments 2-12, wherein the skin sample is maintained at a temperature of 5-37 ° C in a container containing a sterile medium prior to isolating fibroblasts and keratinocytes from the skin sample.

[0065] 14. The method of any of embodiments 2-13, further comprising adding additional sterile medium to the container containing the skin sample and sterile medium prior to isolating fibroblasts and keratinocytes from the skin sample.

[0066] 15. The amount of additional sterile medium is adjusted to fit 1 mm of skin sample. 2 The method of embodiment 14, wherein the amount of the solution is 1 to 10,000 μl per unit volume.

[0067] 16. The method of any of embodiments 2 to 15, wherein fibroblasts are isolated from the skin sample and keratinocytes are isolated by chemical or enzymatic extraction.

[0068] 17. The method of any of embodiments 2 to 15, wherein fibroblasts are separated and keratinocytes are isolated from the skin sample by mechanical extraction.

[0069] 18. The method of embodiment 17, wherein the mechanical extraction comprises shaking a container containing the skin sample and sterile medium.

[0070] 19. The method of embodiment 18, wherein the mechanical extraction comprises agitating the skin sample and sterile medium in the container.

[0071] 20. The method of embodiment 19, wherein the skin sample and sterile medium are agitated in the container for up to 1800 seconds.

[0072] 21. The method of any of embodiments 2-20, further comprising filtering and / or centrifuging the composition comprising fibroblasts and keratinocytes to provide a purified solution of fibroblasts and keratinocytes.

[0073] 22. The method of embodiment 21, wherein the composition comprising fibroblasts and keratinocytes is passed through a filter to provide a purified solution of fibroblasts and keratinocytes.

[0074] 23. The method of embodiment 22, wherein the filter has a pore size of 10 to 100 microns.

[0075] 24. The method of embodiment 21, wherein the composition comprising fibroblasts and keratinocytes is centrifuged to provide a purified solution of fibroblasts and keratinocytes.

[0076] 25. The method of embodiment 24, wherein the composition comprising fibroblasts and keratinocytes is centrifuged at 1 to 300 g.

[0077] 26. The method of embodiment 24 or 25, wherein the composition comprising fibroblasts and keratinocytes is centrifuged for 1 to 60 minutes.

[0078] 27. The method of any of embodiments 24-26, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time.

[0079] 28. The method of embodiment 27, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time at 1 to 300 g.

[0080] 29. The method of embodiment 27 or 28, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time for 1 to 60 minutes.

[0081] 30. The method of any of embodiments 27-29, further comprising removing visible skin debris from the purified solution of fibroblasts and keratinocytes after the first centrifugation and before the second centrifugation.

[0082] 31. The method of embodiment 2, wherein the separation of fibroblasts and keratinocytes from the skin sample results in lysis of all cells of the skin sample.

[0083] 32. The method of any of embodiments 2-31, wherein the matrix has a pH of 6-8.

[0084] 33. The method of embodiment 32, wherein the matrix has a pH of 6.8 to 7.4.

[0085] 34. The method of any of embodiments 2-33, wherein the matrix is ​​a gel matrix.

[0086] 35. The method of embodiment 34, wherein the gel matrix is ​​a collagen solution.

[0087] 36. The method of embodiment 35, wherein the collagen solution is a solution of type I, type II, type III, or type IV collagen.

[0088] 37. The method of embodiment 36, wherein the collagen solution is a solution of type I collagen.

[0089] 38. The method of any of embodiments 2-34, wherein the gel matrix is ​​a keratin solution.

[0090] 39. The method of any of embodiments 2-34, wherein the gel matrix is ​​a fibrin solution.

[0091] 40. The method of any of embodiments 2-34, wherein the gel matrix is ​​a solution comprising collagen, keratin and fibrin.

[0092] 41. The method of any of embodiments 2 to 40, wherein the concentration of the matrix in the matrix containing fibroblasts and keratinocytes is 100 mg / ml or less.

[0093] 42. The method of any of embodiments 2-41, further comprising culturing the matrix containing fibroblasts and keratinocytes.

[0094] 43. The method of embodiment 42, wherein the matrix containing fibroblasts and keratinocytes is cultured for at least 12 hours.

[0095] 44. The method of embodiment 43, wherein the matrix containing fibroblasts and keratinocytes is cultured for 24 to 48 hours.

[0096] 45. The method of any of embodiments 42 to 44, wherein the matrix containing fibroblasts and keratinocytes is cultured at a temperature of 16 to 40 ° C.

[0097] 46. ​​The method of embodiment 45, wherein the matrix containing fibroblasts and keratinocytes is cultured at a temperature of 37°C.

[0098] 47. The method of any of embodiments 42-46, wherein the matrix containing fibroblasts and keratinocytes is cultured in an environment of 0-10% CO2.

[0099] 48. The method of embodiment 47, wherein the matrix containing fibroblasts and keratinocytes is cultured in an environment of 5% CO2.

[0100] 49. A skin graft composition prepared by the method of any of embodiments 2-48.

[0101] 50. A skin graft composition comprising a matrix and a skin sample from which all the cells of the skin sample have been lysed.

[0102] 51. The skin graft composition of embodiment 50, wherein the lysed cells of the skin sample are uniformly dispersed in the matrix.

[0103] 52. A skin graft composition comprising (a) a matrix and (b) fibroblasts and keratinocytes isolated from a skin sample, the composition containing less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblast connective tissue cells per ml.

[0104] 53. The skin graft composition of embodiment 52, wherein the fibroblasts and keratinocytes are uniformly dispersed in the matrix.

[0105] 54. A skin graft composition according to any of embodiments 49 to 53 for use in preparing a skin layer patch or dressing in a subject.

[0106] 55. A method of providing a skin layer patch or dressing to a subject, comprising providing a skin graft composition according to any of the methods of embodiments 2-47, and applying the skin graft composition to a surface of a subject in need of the skin layer patch or dressing.

[0107] 56. The method of embodiment 55, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin layer patch or dressing.

[0108] 57. A method for preparing a skin layer patch or dressing comprising providing a skin graft composition of any of embodiments 49-53 and applying the skin graft composition to a surface.

[0109] 58. The method of embodiment 57, wherein the surface of the subject in need of a skin layer patch or dressing.

[0110] 59. The method of embodiment 58, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin graft.

[0111] 60. The method of embodiment 1, wherein the skin sample is reshaped and resized to obtain a solution comprising the reshaped and resized skin sample, and the solution comprising the reshaped and resized skin sample is combined with a matrix to form a skin graft composition.

[0112] 61. The method of embodiment 60, wherein the skin sample is dissected before being placed in a container with a sterile medium.

[0113] 62. Skin samples must be at least 1 mm 2 62. The method of embodiment 60 or 61, wherein

[0114] 63. The method of any of embodiments 60-62, wherein the container is a glass or plastic container.

[0115] 64. The method of any of embodiments 60-63, wherein the container is a cryovial or a test tube.

[0116] 65. The method of any of embodiments 60-64, wherein the sterile medium is saline.

[0117] 66. The method of embodiment 65, wherein the saline is phosphate buffered saline (PBS).

[0118] 67. The method of any of embodiments 60-66, wherein the sterile medium is Dulbecco's Modified Eagle's Medium (DMEM).

[0119] 68. What volume of sterile medium is needed to hold 1 mm of skin sample? 2 The method of any of embodiments 60 to 67, wherein the amount is 1 to 10,000 μl per unit volume.

[0120] 69. The method of any of embodiments 60-68, wherein the skin sample is maintained in a container with a sterile medium for up to 72 hours prior to reshaping and resizing the skin sample.

[0121] 70. The method of any of embodiments 60-69, wherein prior to reshaping and resizing the skin sample, the skin sample is maintained at a temperature of 5-37 ° C in a container containing a sterile medium.

[0122] 71. The method of any of embodiments 60-70, wherein the method further comprises adding additional sterile medium to the container containing the skin sample and sterile medium prior to reshaping and resizing the skin sample.

[0123] 72. The amount of additional sterile medium is 1 mm 2 The method of embodiment 71, wherein the amount of skin sample is 1 to 10,000 μl.

[0124] 73. The method of any of embodiments 60-72, wherein the skin sample is reshaped and resized by dessication of the skin sample.

[0125] 74. The method of any of embodiments 60-73, wherein the skin sample is removed from the container and sterile medium prior to reshaping and resizing the skin sample, and the container and sterile medium are replaced after reshaping and resizing the skin sample.

[0126] 75. The method of any of embodiments 60-74, further comprising filtering and / or centrifuging the composition containing the reshaped and resized skin sample to provide a purified solution of the reshaped and resized skin sample.

[0127] 76. The method of embodiment 75, wherein the composition containing the reshaped and resized skin sample is passed through a filter to provide a purified solution of the reshaped and resized skin sample.

[0128] 77. The method of embodiment 76, wherein the filter has a pore size of 10 to 100 microns.

[0129] 78. The method of embodiment 76, wherein the composition containing the reshaped and resized skin sample is centrifuged to provide a purified solution of the reshaped and resized skin sample.

[0130] 79. The method of embodiment 78, wherein the composition containing the reshaped and resized skin sample is centrifuged at 1 to 300 g.

[0131] 80. The method of embodiment 78 or 79, wherein the composition containing the reshaped and resized skin sample is centrifuged for 1 to 60 minutes.

[0132] 81. The method of any of embodiments 78-80, wherein the purified solution of the reshaped and resized skin sample is centrifuged at least one additional time.

[0133] 82. The method of embodiment 81, wherein the purified solution of the reshaped and resized skin sample is centrifuged at least one additional time at 1 to 300 g.

[0134] 83. The method of embodiment 81 or 82, wherein the purified solution of the reshaped and resized skin sample is centrifuged at least once additionally for 1 to 60 minutes.

[0135] 84. The method of any of embodiments 60-83, further comprising lysing all cells of the skin sample.

[0136] 85. The method of any of embodiments 60-84, wherein the matrix has a pH of 6-8.

[0137] 86. The method of embodiment 85, wherein the matrix has a pH of 6.8 to 7.4.

[0138] 87. The method of any of embodiments 60-86, wherein the matrix is ​​a gel matrix.

[0139] 88. The method of embodiment 87, wherein the gel matrix is ​​a collagen solution.

[0140] 89. The method of embodiment 88, wherein the collagen solution is a solution of type I, type II, type III, or type IV collagen.

[0141] 90. The method of embodiment 89, wherein the collagen solution is a solution of type I collagen.

[0142] 91. The method of any of embodiments 60-87, wherein the gel matrix is ​​a keratin solution.

[0143] 92. The method of any of embodiments 60-87, wherein the gel matrix is ​​a fibrin solution.

[0144] 93. The method of any of embodiments 60-87, wherein the gel matrix is ​​a solution comprising collagen, keratin and fibrin.

[0145] 94. The method of any of embodiments 60-93, wherein the concentration of the matrix in the matrix containing the reshaped and resized skin sample is 100 mg / ml or less.

[0146] 95. The method of any of embodiments 60-94, wherein the method further comprises culturing a matrix containing the reshaped and resized skin sample.

[0147] 96. The method of embodiment 95, wherein the matrix containing the reshaped and resized skin sample is cultured for at least 12 hours.

[0148] 97. The method of embodiment 96, wherein the matrix containing the reshaped and resized skin sample is cultured for 24 to 48 hours.

[0149] 98. The method of any of embodiments 95 to 97, wherein the matrix containing the reshaped and resized skin sample is cultured at a temperature of 16 to 40°C.

[0150] 99. The method of embodiment 98, wherein the matrix containing the reshaped and resized skin sample is cultured at a temperature of 37°C.

[0151] 100. The method of any of embodiments 95-99, wherein the matrix containing the reshaped and resized skin sample is cultured in an environment of 0-10% CO2.

[0152] 101. The method of embodiment 100, wherein the matrix containing the reshaped and resized skin sample is cultured in an environment of 5% CO2.

[0153] 102. A skin graft composition prepared by the method of any of embodiments 60 to 100.

[0154] 103. A skin graft composition comprising: (a) a matrix; and (b) a reshaped and resized skin sample, the composition containing less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblast connective tissue cells per ml.

[0155] 104. A skin graft composition according to embodiment 102 or 103, for use in preparing a skin layer patch or dressing in a subject.

[0156] 105. A method of providing a skin layer patch or dressing to a subject, comprising providing a skin graft composition according to any of the methods of embodiments 60-100 and applying the skin graft composition to a surface of a subject in need of the skin layer patch or dressing.

[0157] 106. The method of embodiment 105, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin layer patch or dressing.

[0158] 107. A method for preparing a skin layer patch or dressing comprising providing a skin graft composition of embodiment 103 or 104 and applying the skin graft composition to a surface.

[0159] 108. The method of embodiment 107, wherein the skin layer patch or dressing is a surface of a subject requiring the patch or dressing.

[0160] 109. The method of embodiment 108, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin graft. EXAMPLES

[0161] Example 1 This example illustrates the preparation of an exemplary skin graft composition of the invention.

[0162] 5.0 ml of collagen was placed in a sterile tube. 0.37 ml of titration buffer was pipetted into the center of the collagen in the sterile tube. The tube was inverted until thoroughly mixed (approximately 5 times). After 30 minutes, the pH was recorded to ensure that the collagen was neutralized (i.e., at a pH as described herein).

[0163] 1 cm from the patient 2 Skin samples were taken from the post-auricular area (behind the ear) of each mouse. To collect the skin samples, the area was anesthetized with lidocaine and a scalpel was used to remove the skin samples. The skin samples were placed in a vial with sterile PBS and stored at room temperature.

[0164] The skin samples were then processed to prepare the skin graft composition. The skin samples were removed from the vials and the PBS was discarded. The skin samples were washed 10 times with PBS by pipetting. A scalpel was used to dissect the skin samples to obtain the required number of tissue pieces. Four pieces of skin samples, each about 5 mm in diameter, were obtained. If the skin sample had a subcutaneous fat layer, a scalpel was used to carefully remove / scape off the fat.

[0165] Four pieces of skin tissue were placed in the C tube and 1 ml of PBS was added. The C tube was placed in the GENTLEMACS® dissociator and the m_spleen_01 program preprogrammed in the GENTLEMACS® dissociator was run. The m_spleen_01 program was run for 56 seconds, during which the rotator switched direction from clockwise to counterclockwise every 1-2 s and back again. The C tube was then centrifuged at 1200 rpm for 10 minutes. 750 μl of the supernatant was removed from the C tube using a pipette and discarded. The supernatant (including the pellet) remaining in the C tube was 300 μl or less. The pellet containing the fibroblasts and keratinocytes was transferred to the tube containing the neutralized collagen using a pipette to prepare the skin graft composition. The skin graft composition was then stored at ambient conditions (room temperature, i.e., between 15-25°C, humidity between 30-60%, avoiding direct exposure to light).

[0166] Example 2 This example demonstrates the effectiveness of the skin graft composition of the present invention.

[0167] The skin graft composition was prepared as described in Example 1. A physician applied the skin graft composition to the patient's wound and covered the wound with a porous silicone membrane and / or gauze. The silicone membrane and / or gauze were replaced as necessary. Four patients were treated with the skin graft composition of the present invention as follows:

[0168] Patient 1 was 43 years old and had tissue necrosis after cosmetic surgery on the lower abdomen and navel. Patient 2 was 51 years old and had a non-healing wound and ulcers on the ankle and part of the left leg from an accident 5 years ago. Patient 3 was 26 years old and had a healing wound after breast surgery. Patient 3 was taking anticoagulants. Patient 4 had a non-closing wound from breast surgery. Wound measurements were taken before and after a certain number of days after treatment. The wounds of Patients 1 and 2 were measured 30 days after treatment. The wound of Patient 3 was measured 34 days after treatment. The wound of Patient 4 was measured 15 days after treatment. Table 1 reports the wound measurements. Epithelialization indicates wound closure.

[0169] [Table 1]

[0170] Patient 2 also reported a 50% reduction in pain and inflammation. At day 30, the wound was 60% closed. Patient 4 experienced a 12% reduction in the area of ​​Wound 2 (0.51 cm 2 The area of ​​Wound 1 was reduced by 1%. The depths of both Wound 1 and Wound 2 were reduced.

[0171] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0172] The use of the terms "a" and "an" and "the" and "at least one" and similar reference words in the context of describing the present invention (particularly in the context of the claims below) shall be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") shall be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. Reference to a range of values ​​herein is intended to serve merely as a shorthand for referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually referred to herein, unless otherwise indicated herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illuminate the invention, and do not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any element other than any of the claims is essential to the practice of the invention.

[0173] Preferred embodiments of the invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, combinations of the elements described above in all possible variations thereof are encompassed within the invention, whether as described herein or unless otherwise clearly contradicted by context.

Claims

1. A method of preparing a skin graft composition comprising: (a) obtaining a skin sample from a subject in need of a skin layer patch or dressing; (b) placing the skin sample in a container containing a sterile medium; (c) isolating fibroblasts and keratinocytes from the skin sample to obtain a solution comprising fibroblasts and keratinocytes or reshaping and resizing the skin sample to obtain a solution comprising a reshaped and resized skin sample; and (d) combining the solution containing the fibroblasts and keratinocytes, or the solution containing the remodeled and resized skin sample, with the matrix to form a skin graft composition.

2. 2. The method of claim 1, further comprising isolating fibroblasts and keratinocytes from a skin sample to obtain a solution containing fibroblasts and keratinocytes, and combining the solution containing fibroblasts and keratinocytes with a matrix, thereby forming a skin graft composition.

3. 3. The method of claim 2, wherein after isolating the fibroblasts and keratinocytes from the skin sample, the fibroblasts and keratinocytes are viable.

4. 4. The method of claim 2 or 3, wherein the skin sample is dissected prior to placing the skin sample in the container with the sterile medium.

5. Skin samples must be at least 1 mm ２ The method according to any one of claims 2 to 4,

6. The method of any one of claims 2 to 5, wherein the container is a glass container or a plastic container.

7. The method of any one of claims 2 to 5, wherein the container is a cryovial or a test tube.

8. The method of any one of claims 2 to 7, wherein the sterile medium is physiological saline.

9. The method of claim 8, wherein the saline is phosphate buffered saline (PBS).

10. The method of any one of claims 2 to 7, wherein the sterile medium is Dulbecco's Modified Eagle's Medium (DMEM).

11. The amount of sterile medium is 1 mm for each skin sample. ２ The method according to any one of claims 2 to 10, wherein the amount of the solution is 1 to 10,000 μl per unit time.

12. The method of any one of claims 2 to 11, wherein the skin sample is maintained in a container with a sterile medium for up to 72 hours before isolating the fibroblasts and keratinocytes from the skin sample.

13. The method according to any one of claims 2 to 12, wherein the skin sample is maintained at a temperature between 5 and 37°C in a container containing a sterile medium prior to isolating the fibroblasts and keratinocytes from the skin sample.

14. 14. The method of any one of claims 2 to 13, further comprising adding additional sterile medium to the container containing the skin sample and sterile medium prior to isolating fibroblasts and keratinocytes from the skin sample.

15. The amount of additional sterile medium was determined based on the amount of skin sample per mm ２ The method of claim 14, wherein the amount of the solution is 2 to 10,000 μl per unit time.

16. The method according to any one of claims 2 to 15, wherein fibroblasts are isolated and keratinocytes are isolated from the skin sample by chemical or enzymatic extraction.

17. The method according to any one of claims 2 to 15, wherein fibroblasts are isolated and keratinocytes are isolated from the skin sample by mechanical extraction.

18. 20. The method of claim 17, wherein the mechanical extraction comprises shaking a container containing the skin sample and the sterile medium.

19. 20. The method of claim 18, wherein the mechanical extraction comprises agitating the skin sample and the sterile medium in the container.

20. 20. The method of claim 19, wherein the skin sample and sterile medium are agitated in the container for up to 1800 seconds.

21. 21. The method of any one of claims 2 to 20, further comprising filtering and / or centrifuging the composition comprising fibroblasts and keratinocytes to provide a purified solution of fibroblasts and keratinocytes.

22. 22. The method of claim 21, wherein the composition comprising fibroblasts and keratinocytes is passed through a filter to provide a purified solution of fibroblasts and keratinocytes.

23. 23. The method of claim 22, wherein the filter has a pore size of 10 to 100 microns.

24. 22. The method of claim 21, wherein the composition comprising fibroblasts and keratinocytes is centrifuged to provide a purified solution of fibroblasts and keratinocytes.

25. The method of claim 24, wherein the composition comprising fibroblasts and keratinocytes is centrifuged at 1 to 300 g.

26. The method of claim 24 or 25, wherein the composition comprising fibroblasts and keratinocytes is centrifuged for 1 to 60 minutes.

27. The method of any one of claims 24 to 26, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time.

28. 28. The method of claim 27, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time at 1-300 g.

29. The method of claim 27 or 28, wherein the purified solution of fibroblasts and keratinocytes is centrifuged at least one additional time for an additional period of 1 to 60 minutes.

30. 30. The method of any one of claims 27 to 29, further comprising removing visible skin debris from the purified solution of fibroblasts and keratinocytes after the first centrifugation and before the second centrifugation.

31. The method of claim 2, wherein the separation of fibroblasts and keratinocytes from the skin sample results in lysis of all cells of the skin sample.

32. The method of any one of claims 2 to 31, wherein the matrix has a pH of 6 to 8.

33. 33. The method of claim 32, wherein the matrix has a pH of 6.8 to 7.

4.

34. The method of any one of claims 2 to 33, wherein the matrix is ​​a gel matrix.

35. 35. The method of claim 34, wherein the gel matrix is ​​a collagen solution.

36. 36. The method of claim 35, wherein the collagen solution is a solution of type I, type II, type III, or type IV collagen.

37. 37. The method of claim 36, wherein the collagen solution is a solution of type I collagen.

38. The method of any one of claims 2 to 34, wherein the gel matrix is ​​a keratin solution.

39. The method of any one of claims 2 to 34, wherein the gel matrix is ​​a fibrin solution.

40. The method of any one of claims 2 to 34, wherein the gel matrix is ​​a solution comprising collagen, keratin and fibrin.

41. The method of any one of claims 2 to 40, wherein the concentration of matrix in the matrix containing fibroblasts and keratinocytes is 100 mg / ml or less.

42. The method of any one of claims 2 to 41, further comprising culturing the matrix containing the fibroblasts and keratinocytes.

43. 43. The method of claim 42, wherein the matrix containing the fibroblasts and keratinocytes is cultured for at least 12 hours.

44. The method of claim 43, wherein the matrix containing the fibroblasts and keratinocytes is cultured for 24 to 48 hours.

45. The method of any one of claims 42 to 44, wherein the matrix containing the fibroblasts and keratinocytes is cultured at a temperature between 16 and 40°C.

46. 46. ​​The method of claim 45, wherein the matrix containing the fibroblasts and keratinocytes is cultured at a temperature of 37°C.

47. The matrix containing fibroblasts and keratinocytes was incubated at 4°C for 1 h in 0-10% CO ２ The method according to any one of claims 42 to 46, wherein the culture is performed in an environment of

48. The matrix containing fibroblasts and keratinocytes was incubated at 4 °C for 3 min at 5% CO. ２ The method of claim 47, wherein the culture is in an environment of

49. A skin graft composition prepared by the method of any one of claims 2 to 48.

50. A skin graft composition comprising a matrix and a skin sample from which all the cells of the skin sample have been lysed.

51. 51. The skin graft composition of claim 50, wherein the lysed cells of the skin sample are uniformly dispersed throughout the matrix.

52. A skin graft composition comprising (a) a matrix and (b) fibroblasts and keratinocytes isolated from a skin sample, the composition containing less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblast connective tissue cells per ml.

53. 53. The skin graft composition of claim 52, wherein the fibroblasts and keratinocytes are uniformly dispersed throughout the matrix.

54. 54. A skin graft composition according to any one of claims 49 to 53 for use in the preparation of a skin layer patch or dressing in a subject.

55. 48. A method of providing a skin layer patch or dressing to a subject comprising providing a skin graft composition according to the method of any one of claims 2 to 47 and applying the skin graft composition to a surface of a subject in need of the skin layer patch or dressing.

56. 56. The method of claim 55, wherein the skin graft composition is extruded from the press tube onto the surface of a subject in need of a skin layer patch or dressing.

57. 54. A method of preparing a skin layer patch or dressing comprising providing a skin graft composition of any one of claims 49 to 53 and applying the skin graft composition to a surface.

58. 58. The method of claim 57, wherein the skin layer patch or dressing is on the surface of a subject in need thereof.

59. 60. The method of claim 58, wherein the skin graft composition is extruded from the press tube onto the surface of a subject in need of a skin graft.

60. The method of claim 1, further comprising reshaping and resizing a skin sample to obtain a solution containing the reshaped and resized skin sample, and combining the solution containing the reshaped and resized skin sample with a matrix to form a skin graft composition.

61. 61. The method of claim 60, wherein the skin sample is dissected prior to placing the skin sample in the container with the sterile medium.

62. Skin samples must be at least 1 mm ２ 62. The method of claim 60 or 61,

63. 63. The method of any one of claims 60 to 62, wherein the container is a glass or plastic container.

64. 64. The method of any one of claims 60 to 63, wherein the container is a cryovial or a test tube.

65. 65. The method of any one of claims 60 to 64, wherein the sterile medium is saline.

66. 66. The method of claim 65, wherein the saline is phosphate buffered saline (PBS).

67. 67. The method of any one of claims 60 to 66, wherein the sterile medium is Dulbecco's Modified Eagle's Medium (DMEM).

68. The amount of sterile medium is 1 mm for each skin sample. ２ The method of any one of claims 60 to 67, wherein the amount of the solution is 1 to 10,000 μl per unit time.

69. 69. The method of any one of claims 60 to 68, wherein the skin sample is maintained in a container with a sterile medium for up to 72 hours prior to reshaping and resizing the skin sample.

70. 70. The method of any one of claims 60 to 69, wherein prior to reshaping and resizing the skin sample, the skin sample is maintained at a temperature between 5 and 37°C in a container containing a sterile medium.

71. 71. The method of any one of claims 60 to 70, wherein the method further comprises adding additional sterile medium to the container containing the skin sample and sterile medium prior to reshaping and resizing the skin sample.

72. The amount of additional sterile medium is 1 mm ２ 72. The method of claim 71, wherein the volume is between 1 and 10,000 μl per skin sample.

73. The method of any one of claims 60 to 72, wherein the skin sample is reshaped and resized by dessication of the skin sample.

74. 74. The method of any one of claims 60 to 73, wherein prior to reshaping and resizing the skin sample, the skin sample is removed from the container and sterile medium, and after reshaping and resizing the skin sample, the container and sterile medium are replaced.

75. 75. The method of any one of claims 60 to 74, further comprising filtering and / or centrifuging the composition containing the reshaped and resized skin sample to provide a purified solution of the reshaped and resized skin sample.

76. 76. The method of claim 75, wherein the composition containing the reshaped and resized skin sample is passed through a filter to provide a purified solution of the reshaped and resized skin sample.

77. 77. The method of claim 76, wherein the filter has a pore size of 10 to 100 microns.

78. 77. The method of claim 76, wherein the composition containing the reshaped and resized skin sample is centrifuged to provide a purified solution of the reshaped and resized skin sample.

79. 80. The method of claim 78, wherein the composition containing the reshaped and resized skin sample is centrifuged at 1-300 g.

80. 80. The method of claim 78 or 79, wherein the composition containing the reshaped and resized skin sample is centrifuged for 1 to 60 minutes.

81. 81. The method of any one of claims 78 to 80, wherein the purified solution of reshaped and resized skin sample is centrifuged at least one additional time.

82. 82. The method of claim 81, wherein the purified solution of reshaped and resized skin sample is centrifuged at least one additional time at 1-300 g.

83. 83. The method of claim 81 or 82, wherein the purified solution of reshaped and resized skin sample is centrifuged at least one additional time for 1 to 60 minutes.

84. 84. The method of any one of claims 60 to 83, further comprising lysing all cells of the skin sample.

85. The method of any one of claims 60 to 84, wherein the matrix has a pH of 6 to 8.

86. 86. The method of claim 85, wherein the matrix has a pH of 6.8 to 7.

4.

87. The method of any one of claims 60 to 86, wherein the matrix is ​​a gel matrix.

88. 88. The method of claim 87, wherein the gel matrix is ​​a collagen solution.

89. 90. The method of claim 88, wherein the collagen solution is a solution of type I, type II, type III, or type IV collagen.

90. 90. The method of claim 89, wherein the collagen solution is a solution of type I collagen.

91. The method of any one of claims 60 to 87, wherein the gel matrix is ​​a keratin solution.

92. The method of any one of claims 60 to 87, wherein the gel matrix is ​​a fibrin solution.

93. The method of any one of claims 60 to 87, wherein the gel matrix is ​​a solution comprising collagen, keratin and fibrin.

94. 94. The method of any one of claims 60 to 93, wherein the concentration of matrix in the matrix containing the reshaped and resized skin sample is 100 mg / ml or less.

95. 95. The method of any one of claims 60 to 94, further comprising culturing the matrix containing the reshaped and resized skin sample.

96. 96. The method of claim 95, wherein the matrix containing the reshaped and resized skin sample is cultured for at least 12 hours.

97. 97. The method of claim 96, wherein the matrix containing the reshaped and resized skin sample is cultured for 24 to 48 hours.

98. 98. The method of any one of claims 95 to 97, wherein the matrix containing the reshaped and resized skin sample is cultured at a temperature between 16 and 40°C.

99. 99. The method of claim 98, wherein the matrix containing the reshaped and resized skin sample is cultured at a temperature of 37°C.

100. The matrix containing the reshaped and resized skin samples was incubated at 37 °C for 3 min at 0-10% CO ２ The method according to any one of claims 95 to 99, wherein the culture is performed in an environment of

101. The matrices containing the reshaped and resized skin samples were incubated at 4°C for 30 min at 5% CO. ２ The method of claim 100, wherein the culture is in an environment of

102. A skin graft composition prepared by the method of any one of claims 60 to 100.

103. A skin graft composition comprising: (a) a matrix; and (b) a reshaped and resized skin sample, the composition containing less than 10,000 hair follicles, melanocytes, adipocytes, capillaries, and non-fibroblast connective tissue cells per ml.

104. 104. A skin graft composition as described in claim 102 or 103 for use in preparing a skin layer patch or dressing in a subject.

105. A method of providing a skin layer patch or dressing to a subject, the method comprising providing a skin graft composition according to the method of any one of claims 60 to 100 and applying the skin graft composition to a surface of a subject in need of the skin layer patch or dressing.

106. 106. The method of claim 105, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin layer patch or dressing.

107. 105. A method of preparing a skin layer patch or dressing comprising providing the skin graft composition of claim 103 or 104 and applying the skin graft composition to a surface.

108. 108. The method of claim 107, which is on the surface of a subject in need of a skin layer patch or dressing.

109. 109. The method of claim 108, wherein the skin graft composition is extruded from a press tube onto the surface of a subject in need of a skin graft.

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