Wound dressing composition and method of making and using same

Abstract

A method for preparing a wound dressing composition by (a) culturing dermis cells from a cell line, (b) harvesting the dermis cells obtained in step (a) to obtain a crude solution of the dermis cells, (c) centrifuging the crude solution obtained in step (b) to obtain the dermis cells and a supernatant and discarding the supernatant, (d) lysing the dermis cells obtained in step (c) to obtain lysed dermis cells, and (e) combining the lysed dermis cells with a matrix to thereby form a wound dressing composition. A wound dressing composition and methods of providing and preparing a skin layer patch or dressing.

Description

Leydig 7754291WOUND DRESSING COMPOSITION AND METHOD OF MAKING AND USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of US Provisional Application No. 63 / 788,547, filed April 14, 2025, the disclosure of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION

[0002] Chronic wounds represent a significant burden on individuals and healthcare systems worldwide. Approximately 1 in 38 adults annually suffer from chronic wounds. Chronic wounds significantly diminish patients’ quality of life, affecting physical function, psychological well-being, and social interactions.

[0003] Thus, new wound dressing compositions are still needed.BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a method for preparing a wound dressing composition comprising: (a) culturing dermis cells from a cell line, (b) harvesting the dermis cells obtained in step (a) to obtain a crude solution of the dermis cells, (c) centrifuging the crude solution obtained in step (b) to obtain the dermis cells and a supernatant and discarding the supernatant, (d) lysing the dermis cells obtained in step (c) to obtain lysed dermis cells, and (e) combining the lysed dermis cells with a matrix to thereby form a wound dressing composition. The invention also provides a wound dressing composition prepared by the methods described herein.

[0005] The invention provides a wound dressing composition comprising (a) a matrix and (b) lysed and lyophilized sterile dermis cells. The invention also provides a method of providing a skin layer patch or dressing to a subject comprising providing a wound dressing composition in accordance with the methods described herein and applying the wound dressing composition to a surface of the subject that requires a skin layer patch or dressing.

[0006] The invention provides a method of preparing a skin layer patch or dressing comprising providing a wound dressing composition as described herein and applying the wound dressing composition to a surface.Leydig 7754292BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] The figure is a graph of wound area versus study day of long-term wounds treated with saline (Group A, leftmost bar of each group of three), reference treatment (Group B, middle bar of each group of three), or the inventive wound dressing composition (Group C, rightmost bar of each group of three).DETAILED DESCRIPTION OF THE INVENTION

[0008] The inventive wound dressing composition is prepared by a method comprising, consisting essentially of, or consisting of (a) culturing dermis cells from a cell line, (b) harvesting the dermis cells obtained in step (a) to obtain a crude solution of the dermis cells, (c) centrifuging the crude solution obtained in step (b) to obtain the dermis cells and a supernatant and discarding the supernatant, (d) lysing the dermis cells obtained in step (c) to obtain lysed dermis cells, and (e) combining the lysed dermis cells with a matrix to thereby form a wound dressing composition.

[0009] The method comprises culturing dermis cells from a cell line. The dermis cells can be any suitable dermis cells. In embodiments, the dermis cells are fibroblasts, keratinocytes, mesenchymal cells, or adipocytes. In some embodiments, the dermis cells are fibroblasts. In embodiments, the dermis cells are a combination of two or more types of dermis cells. For example, in some embodiments, the dermis cells are fibroblasts and keratinocytes, fibroblasts and mesenchymal cells, fibroblasts and adipocytes, keratinocytes and mesenchymal cells, keratinocytes and adipocytes, or mesenchymal cells and adipocytes. In embodiments, the dermis cells are a combination of three or more types of dermis cells. For example, in some embodiments, the dermis cells are fibroblasts, keratinocytes, and mesenchymal cells; fibroblasts, keratinocytes, and adipocytes; fibroblasts, mesenchymal cells, and adipocytes; or keratinocytes, mesenchymal cells, or adipocytes. In embodiments, the dermis cells are combination of for or more types of dermis cells. For example, in some embodiments, the dermis cells are fibroblasts, keratinocytes, mesenchymal cells, and adipocytes. Any suitable cell line can be used.

[0010] The dermis cells can be cultured by any suitable method of culturing cells using any suitable culture media, including commercially available culture media. In embodiments, the culture media is xeno-free.

[0011] The method comprises harvesting the cultured dermis cells to obtain a crude solution of the dermis cells. The cultured dermis cells can be harvested in any suitableLeydig 7754293manner. In some embodiments, the cultured dermis cells are harvested by dissociation whereby one or more dissociation enzymes is added to the dermis cell culture. Any suitable dissociation enzyme can be used. In some embodiments, the dissociation enzyme is trypsin. In embodiments where the cells are harvested by dissociation, following the addition of a dissociation enzyme, a neutralization solution can be added to the dermis cell culture. Any suitable neutralization solution can be used.

[0012] Any suitable amount of dermis cells can be harvested from culture. In some embodiments, at least one billion dermis cells are harvested. In one embodiment, the one billion dermis cells can be separated into smaller groups of dermis cells for processing to prepare a wound dressing composition. In some embodiments, 5 million - 50 million dermis cells are harvested. In some embodiments, 6 million - 30 million dermis cells are harvested, e.g., 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 17 million, 18 million, 19 million, 20 million, 21 million, 22 million, 23 million, 24 million, 25 million, 26 million, 27 million, 28 million, 29 million, or 30 million, or a number within a range as defined by any two of the foregoing values. In some embodiments, at least 6 million dermis cells are harvested. In some embodiments, 30 million dermis cells are harvested.

[0013] In some embodiments, the harvesting of the dermis cells results in collection of material other than the dermis cells, such as culture media, dissociation enzyme or solution, or other non-dermis cell components. In some embodiments, the harvesting of the dermis cells results in the lysing of some of the dermis cells, which may result in constituent parts of the dermis cells leaving the cell. Such culture media, dissociation enzyme or solution, other non-dermis cell components, or constituent parts from lysed dermis cells together with the dermis cells form a crude solution of the dermis cells.

[0014] In some embodiments, the crude solution comprises 6 million- 30 million harvested dermis cells, e.g., 6 million, 8 million, 10 million, 12 million, 14 million, 16 million, 18 million, 20 million, 22 million, 24 million, 26 million, 28 million, 30 million or a number within a range as defined by any two of the foregoing values. In some embodiments, the crude solution comprises 6 million or more harvested dermis cells. In some embodiments, the crude solution comprises 30 million or more harvested dermis cells.

[0015] In some embodiments, the method comprises centrifuging the harvested dermis cells to obtain dermis cells and a supernatant and discarding the resulting supernatant. In some embodiments, centrifugation of the crude solution of the dermis cells includes centrifugation of the culture media, dissociation enzyme or solution, other non-dermis cellLeydig 7754294components, or constituent parts from lysed dermis cells that are collected together with the dermis cells during harvesting of the dermis cells. The resulting supernatant from centrifugation can comprise the culture media, dissociation enzyme or solution, or other nondermis cell components that may be collected with the dermis cells during harvesting of the dermis cells. In embodiments, the resulting supernatant is discarded following centrifugation.

[0016] The harvested dermis cells can be centrifuged for any suitable amount of time at any suitable force. Desirably, care is taken to not lyse a substantial amount of dermis cells during centrifugation. In some embodiments, the harvested dermis cells are centrifuged for 1-60 minutes. In some embodiments, the harvested dermis cells are 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 foregoing values. In some embodiments, the harvested dermis cells are centrifuged at 1-400 g. In some embodiments, the harvested dermis cells are centrifuged at 1-300 g. In some embodiments, harvested dermis cells are centrifuged at 100-300 g. In some embodiments, the harvested dermis cells are centrifuged at 150-300 g. In some embodiments, the harvested dermis cells are centrifuged at 180-300 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 at a force within a range defined by any two of the foregoing values. In some embodiments, the harvested dermis cells are centrifuged at at least 100 g. In some embodiments, the harvested dermis cells are centrifuged at 300-2500 RPM. In some embodiments, the harvested dermis cells are centrifuged at 500-1500 RPM, e.g., 500 RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM, 1000 RPM, 1100 RPM, 1200 RPM, 1300 RPM, 1400 RPM, or 1400 RPM, or at an RPM within a range defined by any two of the foregoing values.

[0017] In embodiments, the method further comprises adding one or more protease inhibitors to the dermis cells obtained from the crude solution prior to lysing the cells. Any suitable protease inhibitor can be added to the dermis cells prior to lysing in any suitable amount to prevent protein degradation of the dermis cells during and after lysing. In some embodiments, the one or more protease inhibitors is a commercially available combination of protease inhibitors. In some embodiments, phosphate buffer saline is added to the one or more protease inhibitors prior to adding the protease inhibitors to the dermis cells obtained from the crude solution prior to lysing the cells. For example, in some embodiments, a combination of protease inhibitors combined with phosphate buffer saline is added to the dermis cells obtained from the crude solution prior to freezing the cells for the purpose of lysis.Leydig 7754295

[0018] The method comprises lysing the dermis cells obtained from the crude solution of the dermis cells to obtain lysed dermis cells. The dermis cells can be lysed in any suitable manner. In some embodiments, the cells are lysed by a method that does not introduce any external factors to the dermis cells. Any suitable method of lysing that does not introduce any external factors to the dermis cells can be used. In some embodiments, the cells are lysed in a manner that does not degrade the proteins of the cells. Any suitable method of lysing that does not degrade the proteins of the cells may be used. In some embodiments, the cells are lysed by a physical method. In some embodiments, the physical method to lyse the cells is freezing and thawing the dermis cells. In some embodiments, the cells are lysed by sonication. Any suitable method of sonication can be used. In other embodiments, the cells are lysed using an enzyme. Any suitable enzyme can be used. In some embodiments, the enzyme is lyticase.

[0019] Any suitable method of freezing and thawing the dermis cells can be used to lyse the dermis cells. The cells can be frozen at any suitable temperature for any suitable length of time. In some embodiments, the cells are frozen at a temperature between -20 °C and -100 °C, e.g., -20 °C, -25 °C, -30 °C, -35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60°C, -65 °C, -70 °C, -75 °C, -80 °C, -85 °C, -90 °C, -95 °C, or -100 °C, or a temperature within a range defined by any two of the foregoing values. In some embodiments, the cells are frozen at -20° C to -80 °C. In some embodiments, the cells are frozen for a time between 15 minutes and 480 minutes. In some embodiments, the cells are frozen for a time between 60 minutes and 360 minutes, e.g., 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, 195 minutes, 210 minutes, 225 minutes, 240 minutes, 255 minutes, 270 minutes, 285 minutes, 300 minutes, 315 minutes, 330 minutes, 345 minutes, or 360 minutes, or for a length of time within a range defined by any two of the foregoing values. In some embodiments the cells are frozen for 120 minutes (two hours). The cells can be thawed at any suitable temperature for any suitable length of time. In some embodiments, the cells are thawed at room temperature. In embodiments, room temperature is 20 °C to 25 °C.

[0020] In embodiments, all, or at least substantially all or a significant portion, of the dermis cells are lysed by the lysing process. In embodiments, at least 70% of the dermis cells are lysed, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the dermis cells, or a percentage of dermis cells within a range defined by any two of the foregoing values, are lysed. In embodiments, all, or at least substantially, of the dermis cells are dead.Leydig 7754296

[0021] In embodiments, the method further comprises adding one or more protease inhibitors to the lysed dermis cells after lysing the cells. Any suitable protease inhibitor can be added to the lysed dermis cells after lysing in any suitable amount to prevent protein degradation of the dermis cells after lysing. In some embodiments, the one or more protease inhibitors is a commercially available combination of protease inhibitors. In some embodiments, phosphate buffer saline is added to the one or more protease inhibitors prior to adding the protease inhibitors to the lysed dermis cells after lysing the cells. For example, in some embodiments, a combination of protease inhibitors combined with phosphate buffer saline is added to the lysed dermis cells after freezing the cells for the purpose of lysis. In embodiments, one or more protease inhibitors is added to the dermis cells obtained from the crude solution prior to lysing and to the lysed dermis cells after lysing the cells.

[0022] In embodiments, the method further comprises centrifuging the lysed dermis cells. The lysed dermis cells can be centrifuged for any suitable amount of time at any suitable force. In some embodiments, the lysed dermis cells are centrifuged for 1-60 minutes. In some embodiments, the lysed dermis cells are 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 foregoing values. In some embodiments, the lysed dermis cells are centrifuged at 1-400 g. In some embodiments, the lysed dermis cells are centrifuged at 1-300 g. In some embodiments, the lysed dermis cells are centrifuged at 50-200 g. In some embodiments, the lysed dermis cells are centrifuged at 100-200 g, e.g., 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, or at a force within a range defined by any two of the foregoing values. In some embodiments, the harvested dermis cells are centrifuged at at least 100 g. In some embodiments, the harvested dermis cells are centrifuged at 300-2500 RPM. In some embodiments, the harvested dermis cells are centrifuged at 500-1500 RPM, e.g., 500 RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM, 1000 RPM, 1100 RPM, 1200 RPM, 1300 RPM, 1400 RPM, or 1400 RPM, or at an RPM within a range defined by any two of the foregoing values.

[0023] The method comprises combining the lysed dermis cells with a matrix to form a wound dressing composition. The lysed dermis cells can be combined with a matrix in any suitable manner. In embodiments, the lysed dermis cells are mixed with the matrix. In embodiments, the lysed dermis cells are added onto a surface of a matrix.

[0024] The matrix can be any suitable matrix. In some embodiments, the matrix is a foam, liquid, gel, or solid matrix. In some embodiments, the matrix comprises, consistsLeydig 7754297essentially of, or consists of collagen, fibrin and / or keratin. In some embodiments, the matrix comprises, consists essentially of, or consists of collagen and fibrin. In some embodiments, the matrix comprises, consists essentially of, or consists of collagen and keratin. In some embodiments, the matrix comprises, consists essentially of, or consists of fibrin and keratin. In some embodiments, the matrix comprises, consists essentially of, or consists of collagen, fibrin, and keratin. In some embodiments, the matrix does not comprise any components other than the recited components.

[0025] In some embodiments, the matrix conforms to the surface to which the wound dressing composition is applied. For example, in some embodiments, the matrix conforms to a 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 (GAG), such as hyaluronic acid (HA), a synthetic polymeric fiber made of a poly-acid such as polyactic, polyglycolic or polyamino acid, polycaprolactone, polyamino acid, polypeptide gel, copolymers thereof, each 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 the recited components.

[0026] In some embodiments, the matrix is a solid matrix. In some embodiments, the solid matrix is a powder or sponge. In some embodiments, the matrix is a sponge. In some embodiments, the matrix is a collagen, keratin, or fibrin sponge. In some embodiments the sponge comprises, consists essentially of, or consists of collagen, keratin, or fibrin. In some embodiments the sponge comprises, consists essentially of, or consists of collagen and keratin. In some embodiments the sponge comprises, consists essentially of, or consists ofLeydig 7754298collagen and fibrin. In some embodiments the sponge comprises, consists essentially of, or consists of keratin and fibrin. In some embodiments the sponge comprises, consists essentially of, or consists of collagen, keratin, and fibrin. In some embodiments the matrix is a collagen and keratin sponge, a collagen and fibrin sponge, a keratin and fibrin sponge, or a collagen, keratin, and fibrin sponge. In some embodiments, the matrix does not comprise any components other than the recited components.

[0027] In some embodiments, the matrix has a viscosity of 0.5-1000000 cP. In some embodiments, the matrix has a viscosity of 2000-250000 cP. In some embodiments, the matrix has a viscosity of 10000-100000 cP, e.g., 10000 cP, 15000 cP, 20000 cP, 25000 cP, 30000 cP, 35000 cP, 40000 cP, 45000 cP, 50000 cP, 55000 cP, 60000 cP, 65000 cP, 70000 cP, 7500 cP 0, 80000 cP, 85000 cP, 90000 cP, 95000 cP, or 100000 cP, or a viscosity within a range defined by any two of the foregoing values.

[0028] In some embodiments, the method further comprises lyophilizing and sterilizing the lysed dermis cells and / or the wound dressing composition. Any suitable methods of lyophilizing and sterilizing may be used. In some embodiments, the methods of lyophilizing and sterilizing do not degrade the lysed dermis cells and / or the wound dressing composition. In some embodiments, sterilizing comprises radiation, filtration, or supercritical carbon dioxide sterilization. In some embodiments, radiation comprises e-beaming, X-ray, or gamma radiation.

[0029] In embodiments, the method comprises lyophilizing and sterilizing only the lysed dermis cells. In other embodiments, the method comprises lyophilizing and sterilizing the wound dressing composition. In some embodiments, the method comprises lyophilizing and sterilizing the lysed dermis cells and the wound dressing composition. Lyophilization does not necessarily occur before sterilization, and sterilization does not necessarily occur before lyophilization. In some embodiments, the method comprises combining the lysed dermis cells with a matrix to form a wound dressing composition, then sterilizing the wound dressing composition, and then lyophilizing the wound dressing composition. In other embodiments, the method comprises combining the lysed dermis cells with a matrix to form a wound dressing composition, lyophilizing the wound dressing composition, and then sterilizing the wound dressing composition.

[0030] In some embodiments, lyophilizing of the wound dressing composition results in a bead or beads that can be administered to a patient. In some embodiments, the wound dressing composition is administered as a bead or beads. In some embodiments, the beadsLeydig 7754299are administered directly to a subject in need of a wound dressing composition, e.g., the beads are administered directly to a wound on a subject.

[0031] The invention provides a wound dressing composition. In an embodiment, the wound dressing composition is prepared by any of the methods described herein. In embodiments, the lysed dermis cells are uniformly dispersed in the matrix.

[0032] The wound dressing composition can have any suitable pH. In some embodiments, the wound dressing composition has a pH of 2-8. In some embodiments, the wound dressing composition has a pH of 2-7.4, e.g., pH of 2, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4., 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, or a pH within a range defined by any two of the foregoing values.

[0033] In yet another embodiment, the wound dressing composition comprises (a) a matrix and (b) lysed and lyophilized sterile dermis cells. The matrix and lysed and lyophilized sterile dermis cells are as described herein with respect to other embodiments. In embodiments, the lysed and lyophilized sterile dermis cells are uniformly dispersed in the matrix. In embodiments, the lysed and lyophilized sterile dermis cells are dispersed on top of a surface of the matrix.

[0034] In embodiments, the wound dressing composition is stable, e.g., the wound dressing composition remains effective for its intended use after storage.

[0035] In embodiments, the wound dressing composition is stable after storage at 20°C to 25 °C for one month or more, two months or more, three months or more, six months or more, nine months or more, one year or more, two years or more, three years or more, four years or more, or five years or more. In embodiments, the wound dressing composition is stable after storage at 2 °C to 4 °C for one month or more, two months or more, three months or more, six months or more, nine months or more, one year or more, two years or more, three years or more, four years or more, or five years or more. In embodiments, the wound dressing composition is stable after storage at -20 °C to -80 °C for one month or more, two months or more, three months or more, six months or more, nine months or more, one year or more, two years or more, three years or more, four years or more, or five years or more.

[0036] In embodiments, the stability of the wound dressing composition is determined by the protein content of the wound dressing composition at the end of one of the aforementioned times. In embodiments, the wound dressing composition is considered stable after a particular amount of time if the wound dressing composition at the end of the particular amount of time comprises at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atLeydig 77542910least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the protein content of the wound dressing composition upon preparation. In embodiments, the stability of the wound dressing composition is determined by the matrix content of the wound dressing composition at the end of one of the aforementioned times. In embodiments, the wound dressing composition is considered stable after a particular amount of time if the wound dressing composition at the end of the particular amount of time comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, of the matrix content of the wound dressing composition upon preparation.

[0037] The wound dressing composition can be stored at any suitable temperature. In embodiments, the wound dressing composition is stored at room temperature or at a temperature between 2 and 4 °C. In embodiments, the wound dressing composition is stored at room temperature. In embodiments, room temperature is 20 °C to 25 °C, e.g., 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, or 25 °C, or at a temperature within a range defined by any two of the foregoing values. In embodiments, the wound dressing composition is stored at a temperature between 2 and 4 °C, e.g., 2 °C, 2.5 °C, 3 °C, 3.5 °C, or 4 °C, or at a temperature within a range defined by any two of the foregoing values. In other embodiments, the wound dressing composition is stored at 0 °C or below, e.g., at -10 °C or below, at -20 °C or below, -40 °C or below, -60 °C or below, such as at -80 °C.

[0038] The wound dressing composition provided by the invention can be for use in the preparation of a skin layer patch or dressing for a subject. The invention provides a method of providing a skin layer patch or dressing to a subject comprising providing a wound dressing composition as described herein and / or prepared by a method as described herein, and applying the wound dressing composition to the surface of the subject that requires a skin layer patch or dressing, e.g., a subject with a wound to which the wound dressing composition can be suitably applied.

[0039] The invention provides a method of preparing a skin layer patch or dressing comprising providing a wound dressing composition as described herein, and applying the wound dressing composition to any suitable surface. In some embodiments, the surface on a subject is a surface in need of coverage and protection that would typically be provided by healthy skin. For example, the surface on a subject can 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.Leydig 77542911

[0040] In embodiments of the invention, the wound dressing composition is applied to the surface of a subject that requires a skin layer patch or dressing, in any suitable manner and by any suitable means. For example, the wound dressing 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 with a plunger. In some embodiments, the press tube is a pouch. In other embodiments, the wound dressing composition is applied by spreading the wound dressing composition onto the surface of the subject using any suitable tool, such as a spatula. In other embodiments, the wound dressing composition is applied by pouring the wound dressing composition onto the surface of the subject. In other embodiments the wound dressing composition is sprayed onto the surface of the subject. In other embodiments, the wound dressing composition is distributed by gravity onto the surface of the subject.

[0041] In other embodiments, the matrix is applied to the surface of a subject that requires a skin layer patch or dressing, such as a wound, and then the lysed dermis cells are applied to the top of the matrix. In embodiments, the matrix is a solid dry pad.

[0042] Embodiments

[0043] 1. A method for preparing a wound dressing composition comprising: (a) culturing dermis cells from a cell line, (b) harvesting the dermis cells obtained in step (a) to obtain a crude solution of the dermis cells, (c) centrifuging the crude solution obtained in step (b) to obtain the dermis cells and a supernatant and discarding the supernatant, (d) lysing the dermis cells obtained in step (c) to obtain lysed dermis cells, and (d) combining the lysed dermis cells with a matrix to thereby form a wound dressing composition.

[0044] 2. The method of embodiment 1, wherein the dermis cells are fibroblasts, keratinocytes, mesenchymal cells and / or adipocytes.

[0045] 3. The method of embodiment 1 or 2, wherein the dermis cells are fibroblasts.

[0046] 4. The method of any one of embodiments 1-3, wherein the crude solution comprises at least 6 million dermis cells.

[0047] 5. The method of any one of embodiments 1-4, wherein the crude solution obtained in step (b) is centrifuged at least once.

[0048] 6. The method of any one of embodiments 1-5, wherein the crude solution obtained in step (b) is centrifuged at least at 1000 RPM and / or or at least at 100g.

[0049] 7. The method of any one of embodiments 1-6, wherein one or more protease inhibitors is added to the dermis cells obtained from the crude solution prior to lysing.Leydig 77542912

[0050] 8. The method of any one of embodiments 1-7, wherein the dermis cells obtained in step (c) are lysed by a method that does not introduce any external factors.

[0051] 9. The method of embodiment 8, wherein the method that does not introduce any external factors is a physical method.

[0052] 10. The method of embodiment 9, wherein the physical method is freezing and thawing the dermis cells.

[0053] 11. The method of embodiment 10, wherein the dermis cells are frozen at -20 °C to -80 °C for 2 hours or more.

[0054] 12. The method of any one of embodiments 1-11, wherein one or more protease inhibitors is added to the lysed cells.

[0055] 13. The method of any one of embodiments 1-12, wherein the method further comprises lyophilizing and sterilizing the lysed dermis cells obtained in step (d).

[0056] 14. The method of embodiment 13, wherein sterilizing comprises radiation, filtration, or supercritical carbon dioxide sterilization.

[0057] 15. The method of embodiment 14, wherein sterilizing comprises radiation, and wherein the radiation comprises e-beaming, X-ray, or gamma radiation.

[0058] 16. The method of any one of embodiments 1-15, wherein the matrix is a foam, liquid, gel, or solid matrix.

[0059] 17. The method of embodiment 16, wherein the matrix is a gel matrix.

[0060] 18. The method of embodiment 17, wherein the gel matrix is a collagen solution.

[0061] 19. The method of embodiment 18, wherein the collagen solution is a solution of type I collagen.

[0062] 20. The method of embodiment 18, wherein the collagen solution is a solution of type II, type III, or type IV collagen.

[0063] 21. The method of embodiment 17, wherein the gel matrix is a keratin solution.

[0064] 22. The method of embodiment 17, wherein the gel matrix is a fibrin solution.

[0065] 23. The method of embodiment 17, wherein the gel matrix is a solution comprising collagen, keratin, and fibrin.

[0066] 24. The method of embodiment 16, wherein the matrix is a solid matrix.

[0067] 25. The method of embodiment 24, wherein the solid matrix is a powder and / or sponge.

[0068] 26. The method of embodiment 25, wherein the matrix is a collagen, keratin, and / or fibrin sponge.Leydig 77542913

[0069] 27. The method of any one of embodiments 1-26, wherein the method further comprises lyophilizing the wound dressing composition obtained in step (e).

[0070] 28. The method of embodiment 27, wherein the lyophilized wound dressing composition is administered as a bead.

[0071] 29. The method of any one of embodiments 1-28, wherein the method further comprises sterilizing the wound dressing composition.

[0072] 30. The method of embodiment 29, wherein sterilizing comprises radiation, filtration, or supercritical carbon dioxide sterilization.

[0073] 31. The method of embodiment 30, wherein sterilization comprises radiation, and wherein the radiation comprises e-beaming, X-ray, or gamma radiation.

[0074] 32. A wound dressing composition prepared by the method of any one of embodiments 1-31.

[0075] 33. The wound dressing composition of embodiment 32, in which the lysed dermis cells are uniformly dispersed in the matrix.

[0076] 34. The wound dressing composition of embodiments 32 or 33, wherein the wound dressing composition has a pH of 2-8.

[0077] 35. The wound dressing composition of embodiment 34, wherein the wound dressing composition has a pH of 2-7.4.

[0078] 36. A wound dressing composition comprising (a) a matrix and (b) lysed and lyophilized sterile dermis cells.

[0079] 37. The wound dressing composition of embodiment 36, in which the lysed and lyophilized sterile dermis cells are uniformly dispersed in the matrix.

[0080] 38. The wound dressing composition of any of embodiments 34-37, wherein the wound dressing composition is stable after storage at 25 °C for 5 years.

[0081] 39. The wound dressing composition of any of embodiments 34-37, wherein the wound dressing composition is stable after storage at room temperature or at a temperature between 2 and 4 °C for 5 years.

[0082] 40. A wound dressing composition of any one of embodiments 34-38 for use in the preparation of a skin layer patch or dressing for a subject.

[0083] 41. A method of providing a skin layer patch or dressing to a subject comprising providing a wound dressing composition in accordance with the method of any one of embodiments 1-33 and applying the wound dressing composition to a surface of the subject that requires a skin layer patch or dressing.Leydig 77542914

[0084] 42. The method of embodiment 41, wherein the wound dressing composition is extruded from a press tube, extracted from a vial, or sprayed onto the surface on the subject that requires a skin layer patch or dressing.

[0085] 43. The method of embodiment 41, wherein the wound dressing composition is distributed by gravity onto the surface of the subject that requires a skin layer patch or dressing.

[0086] 44. A method of preparing a skin layer patch or dressing comprising providing a wound dressing composition of any one of embodiments 34-40 and applying the wound dressing composition to a surface.

[0087] 45. The method of embodiment 44, wherein a surface on the subject requires a skin layer patch or dressing.

[0088] 46. The method of embodiment 45, wherein the wound dressing composition is extruded from a press tube or extracted from a vial onto the surface on the subject that requires a skin layer patch or dressing.

[0089] 47. The method of embodiment 45, wherein the wound dressing composition is distributed by gravity onto the surface of the subject that requires a skin layer patch or dressing.

[0090] The following example further illustrates the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLE

[0091] This example demonstrates use of the inventive wound dressing composition.

[0092] A total of 25 Gottingen minipigs (13 male and 12 female) were randomly assigned to three study groups. The study groups included two treatment groups and one control group. Each animal had three sets of wounds created on the back of the animal on Day 0 (long-term wounds), Day 11 (mid-term wounds), and Day 18 (short-term wounds) with a scalpel. The wounds were approximately 1.5 ± .1 cm x 1.5 ± .1 cm with a depth removing epidermis, dermis, and connective tissue (approximately 0.8-1 cm).

[0093] Animals in the control group received topical administration of 0.5 mL of- 0.5 mL of saline solution per wound (Group A). Animals in the first treatment group received topical administration of 0.3-0.4 mL of reference product (OCM™ wound matrix by Omeza,Leydig 77542915LLC) per wound (Group B). Animals in the second treatment group received topical administration of ~ 0.8 mL of the inventive wound dressing (a collagen gel matrix combined with sterilized, lyophilized, lysed fibroblast cells) per wound (Group C). The treatments completely covered the wound surface.

[0094] After applying the treatments, a foam bandage (Animal Polster) was placed over each wound site to protect the wounds from external contact. Additionally, an individual dressing (TEGADERM™ transparent film dressing by 3M Company, Inc.) was attached directly to the skin surrounding the wounds, and the area was covered with a plaster (OMNIFIX™ elastic dressing by Paul Hartmann Spa). An additional bandage (TUBILAST™ elastic bandage by Laboratorios Herbitas S.L.) was used to cover the backs of the animals.

[0095] Long-term wounds were revised on Day 7 ± 1, Day 11, Day 18, and Day 25. Mid-term wounds were revised on Day 11, Day 18, and Day 25. Short-term wounds were revised on Day 18 and Day 25. At each revision, the bandages were removed, a scoring system measuring the presence of exudate, scab formation, presence of edema at the borders, presence of infection, and wound size was applied, and the topical treatment and bandages were reapplied to the wounds.

[0096] Short-term wound healing trends showed no significant differences across Groups A, B, and C by seven days post-wound creation. In the mid-term wounds, 7 days postwound, Group A showed the smallest wound areas (1.3±0.3 cm2), compared to Groups B (1.6±0.2 cm2) and C (1.6±0.3 cm2). The differences between Groups A and C were statistically significant and could be attributed to greater skin shrinkage due to skin dryness of the saline-treated control group. By 14 days post-wound, there were no statistically significant differences across groups, with Group A having the smallest wound areas, then Group C and Group B.

[0097] Long-term healing progress showed that areas treated with the inventive wound dressing composition (Group C) were significantly smaller as compared to wounds treated with the reference treatment (Group B) or saline (Group A). The long-term wound healing progress results for Groups A, B, and C are presented in the figure.

[0098] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.Leydig 77542916

[0099] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to 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” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0100] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to 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, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 77542917CLAIM(S):

1. A method for preparing a wound dressing composition comprising:(a) culturing dermis cells from a cell line,(b) harvesting the dermis cells obtained in step (a) to obtain a crude solution of the dermis cells,(c) centrifuging the crude solution obtained in step (b) to obtain dermis cells and a supernatant and discarding the supernatant,(d) lysing the dermis cells obtained in step (c) to obtain lysed dermis cells, and (e) combining the lysed dermis cells with a matrix to thereby form a wound dressing composition.

2. The method of claim 1, wherein the dermis cells are fibroblasts, keratinocytes, mesenchymal cells and / or adipocytes.

3. The method of claim 1 or 2, wherein the dermis cells are fibroblasts.

4. The method of any one of claims 1-3, wherein the crude solution comprises at least 6 million dermis cells.

5. The method of any one of claims 1-4, wherein the crude solution obtained in step (b) is centrifuged at least once.

6. The method of any one of claims 1-5, wherein the crude solution obtained in step (b) is centrifuged at least at 1000 RPM and / or or at least at 100g.

7. The method of any one of claims 1-6, wherein one or more protease inhibitors is added to the dermis cells obtained from the crude solution prior to lysing.

8. The method of any one of claims 1-7, wherein the dermis cells obtained in step (c) are lysed by a method that does not introduce any external factors.

9. The method of claim 8, wherein the method that does not introduce any external factors is a physical method.

10. The method of claim 9, wherein the physical method is freezing and thawing the dermis cells.Leydig 7754291811. The method of claim 10, wherein the dermis cells are frozen at -20 °C to -80 °C for 2 hours or more.

12. The method of any one of claims 1-11, wherein one or more protease inhibitors is added to the lysed cells.

13. The method of any one of claims 1-12, wherein the method further comprises lyophilizing and sterilizing the lysed dermis cells obtained in step (d).

14. The method of claim 13, wherein sterilizing comprises radiation, filtration, or supercritical carbon dioxide sterilization.

15. The method of claim 14, wherein sterilizing comprises radiation, and wherein the radiation comprises e-beaming, X-ray, or gamma radiation.

16. The method of any one of claims 1-15, wherein the matrix is a foam, liquid, gel, or solid matrix.

17. The method of claim 16, wherein the matrix is a gel matrix.

18. The method of claim 17, wherein the gel matrix is a collagen solution.

19. The method of claim 18, wherein the collagen solution is a solution of type I collagen.

20. The method of claim 18, wherein the collagen solution is a solution of type II, type III, or type IV collagen.

21. The method of claim 17, wherein the gel matrix is a keratin solution.

22. The method of claim 17, wherein the gel matrix is a fibrin solution.

23. The method of claim 17, wherein the gel matrix is a solution comprising collagen, keratin, and fibrin.

24. The method of claim 16, wherein the matrix is a solid matrix.

25. The method of claim 24, wherein the solid matrix is a powder and / or sponge.Leydig 7754291926. The method of claim 25, wherein the matrix is a collagen, keratin, and / or fibrin sponge.

27. The method of any one of claims 1-26, wherein the method further comprises lyophilizing the wound dressing composition obtained in step (e).

28. The method of claim 27, wherein the lyophilized wound dressing composition is administered as a bead.

29. The method of any one of claims 1-28, wherein the method further comprises sterilizing the wound dressing composition.

30. The method of claim 29, wherein sterilizing comprises radiation, filtration, or supercritical carbon dioxide sterilization.

31. The method of claim 30, wherein sterilization comprises radiation, and wherein the radiation comprises e-beaming, X-ray, or gamma radiation.

32. A wound dressing composition prepared by the method of any one of claims 1-31.

33. The wound dressing composition of claim 32, in which the lysed dermis cells are uniformly dispersed in the matrix.

34. The wound dressing composition of claims 32 or 33, wherein the wound dressing composition has a pH of 2-8.

35. The wound dressing composition of claim 34, wherein the wound dressing composition has a pH of 2-7.4.

36. A wound dressing composition comprising (a) a matrix and (b) lysed and lyophilized sterile dermis cells.

37. The wound dressing composition of claim 36, in which the lysed and lyophilized sterile dermis cells are uniformly dispersed in the matrix.

38. The wound dressing composition of any of claims 34-37, wherein the wound dressing composition is stable after storage at 25 °C for 5 years.Leydig 7754292039. The wound dressing composition of any of claims 34-37, wherein the wound dressing composition is stable after storage at room temperature or at a temperature between 2 and 4 °C for 5 years.

40. A wound dressing composition of any one of claims 34-38 for use in the preparation of a skin layer patch or dressing for a subject.

41. A method of providing a skin layer patch or dressing to a subject comprising providing a wound dressing composition in accordance with the method of any one of claims 1-33 and applying the wound dressing composition to a surface of the subject that requires a skin layer patch or dressing.

42. The method of claim 41, wherein the wound dressing composition is extruded from a press tube, extracted from a vial, or sprayed onto the surface on the subject that requires a skin layer patch or dressing.

43. The method of claim 41, wherein the wound dressing composition is distributed by gravity onto the surface of the subject that requires a skin layer patch or dressing.

44. A method of preparing a skin layer patch or dressing comprising providing a wound dressing composition of any one of claims 34-40 and applying the wound dressing composition to a surface.

45. The method of claim 44, wherein a surface on the subject requires a skin layer patch or dressing.

46. The method of claim 45, wherein the wound dressing composition is extruded from a press tube or extracted from a vial onto the surface on the subject that requires a skin layer patch or dressing.

47. The method of claim 45, wherein the wound dressing composition is distributed by gravity onto the surface of the subject that requires a skin layer patch or dressing.

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