Therapeutic use of fibroblasts for use in wound healing

Abstract

To provide the use of activated or inactivated fibroblasts or fibroblast-derived materials, such as exosomes from fibroblasts, for wound care.SOLUTION: A method and a composition are useful for purpose of tissue differentiation, cell recruitment, differentiation of local stem cells, and expansion of local cells, such as keratocytes, vasal epithelial cells, myofibroblasts, and / or dermal fibroblasts, including for initiating and / or maintaining wound healing.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 320,388, filed on March 16, 2022, U.S. Provisional Patent Application Serial No. 63 / 379,220, filed on October 12, 2022, and U.S. Provisional Patent Application Serial No. 63 / 383,498, filed on November 13, 2022, all of which are hereby incorporated by reference in their entirety.

[0002] Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, immunology, and medicine.

Background Art

[0003] Fibroblasts are no longer merely structural components of organs but are considered dynamic participants in multiple systems, such as the complex interactions of multiple systems involved in the immune system and wound healing.

[0004] The skin is the largest organ of the human body, accounting for approximately 15% of body weight, and serves multiple complex functions as a protector from physical, chemical, and microbial external influences, a maintainer of temperature and electrolyte balance, a biological factory for the synthesis and metabolism of structural proteins, lipids, glycans, and signaling molecules, and an essential component of the immune, nervous, and endocrine systems [1]. Thus, skin injury and the skin repair process are well-regulated processes involving multiple systems and cell types. Wound healing follows a complex orchestrated process of hemostasis, inflammation, proliferation, epithelialization, and remodeling limited to the site of injury [2].

[0005] Treating wounds places a significant burden on the healthcare system and disrupts the quality of life for victims of wounds that do not heal. Wounds that are difficult to heal, including chronic wounds, require ongoing, time-consuming, and costly treatment. Furthermore, skin injuries resulting from illnesses (such as diabetes and cancer), cuts, abrasions, bedsores, and burns can have lasting effects on a patient's physical, emotional, and psychological well-being. While some skin wounds heal normally and quickly, underlying health factors such as age, health status, and certain illnesses can negatively impact the complex wound healing process, potentially requiring external intervention for proper healing. [Overview of the project]

[0006] Embodiments of the present disclosure encompass compositions, methods, and systems for introducing activated or inactivated fibroblasts and / or fibroblast-derived materials (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, vesicles, apoptotic bodies, or combinations thereof, or any other fragments or biological components of fibroblasts) onto or into a wound, together with or without other types of cells such as keratinocytes and / or epithelial cells. Cells and / or cell-derived products (including cells and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) can be used on a wound site in any suitable form, including, in some embodiments, 3D cultured cells and / or 3D printed matrices, comprising one or more cell types, by mechanical application, on a device, contained within a gel matrix, or incorporated within a synthetic or organic polymer matrix, at least as a spray, gel, or suspension. In some cases, one or more additional agents, such as one or more growth factors, one or more cytokines, one or more chemokines, and / or one or more activators, may be administered to, around, and / or within the surrounding tissue. In such cases, the additional agent(s) may or may not be provided to the wound(s) in the same formulation or manner as activated or inactivated fibroblasts and / or fibroblast-derived substances (fibroblasts; fibroblast-like cells; and / or extracellular vesicles including exosomes, microvesicles, apoptotic bodies, fibroblast lysates, or combinations thereof, or other fragments or biological components of fibroblasts).

[0007] In certain embodiments, the Disclosure relates to or encompasses interactions between a first type of cell or cell derivative (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, fibroblast lysates, or any other fragments or biological components of fibroblasts) and a second type of cell and / or a specific agent(s), and includes modifications(s) of the first and / or second type of cell as a result of such interactions. In addition, in certain embodiments, the Disclosure includes compositions, methods, and systems in which fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are modified by exposure to one or a combination of specific cells and / or specific agents(s), such as nucleic acids, cytokines, chemokines, and / or growth factors. In certain embodiments, these modified or unmodified fibroblasts (fibroblasts; fibroblast-like cells, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts) can be introduced into and / or on the wound and / or surrounding tissue to initiate processes of proliferation, epithelialization, and induction of other biological processes, contributing to healing necessary for healing and remodeling. For example, regeneration is achieved by regenerating necessary functional cells, including myofibroblasts, vascular epithelial cells, and human dermal fibroblasts, and / or by regenerating extracellular matrix materials such as collagen I-XIV, fibrin, fibronectin, vitronectin, elastin, hyaluronan, chondroitin sulfate, decorin, versican, SPARC, and / or tenacin[3].

[0008] In certain embodiments, the regeneration of these cells and cellular components at the wound site may be carried out through one or more possible steps, such as the following:

[0009] (1) Injecting modified or unmodified fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) into the wound site[4] or applying them topically to the wound and surrounding tissue.

[0010] (2) Recruiting neutrophils, macrophages and platelets to a wound by fibroblasts (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) [5].

[0011] (3) Activating the patient's aged human dermal fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts), keratinocytes, vascular epithelial cells, or myofibroblasts through intercellular (paracrine) contact with modified or unmodified fibroblasts[6] (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts).

[0012] (4) Differentiation of local stem cells, human dermal fibroblasts (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or other fragments or biological components of fibroblasts) by direct or indirect action of the introduced modified / unmodified fibroblasts or the excreted products of modified / unmodified fibroblasts.

[0013] (5) Expanding endogenous human dermal fibroblasts (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing fibroblast exosomes, microvesicles, apoptotic bodies or other fragments or biological components, as well as aged fibroblasts of the individual itself), keratinocytes, vascular epithelial cells, or myofibroblasts remaining in the injured skin to enable them to enter the wound healing process.

[0014] (6) Initiation of angiogenesis by direct and / or indirect interactions of introduced fibroblasts and / or fibroblast secretions[7].

[0015] (7) Recruiting stem cells and any other cells useful in the healing process to the site of injury by direct and / or indirect action of introduced fibroblasts and / or substances excreted by introduced fibroblasts[8].

[0016] In certain embodiments, the disclosure relates to compositions, methods and systems in which specific cells are modified by exposure to specific agents exocrines, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts and / or fibroblasts, or to specific agents excreted from or contained within fibroblasts. In certain embodiments, the interaction between fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles exocrines, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) and one or more other types of cells (and optionally their interaction also includes one or more specific agents) results in modification of fibroblasts and / or modification of other types of cells involved in wound healing. In certain embodiments, the other types of cells include at least recruited immune cells (e.g., dendritic cells, neutrophils, monocytes, macrophages, NK cells, T cells, mast cells, and sin-resident T cells) and local stem cells [8].

[0017] In certain embodiments, fibroblasts and other cells present in the wound and / or surrounding tissue are modified to stimulate wound repair by introducing living single-cell suspensions, living multicellular suspensions, living 3D spheroid fibroblasts, inactivated fibroblasts, dead fibroblasts, and / or fibroblast-derived materials into a wound. In certain aspects, prior or current exposure to fibroblasts and / or fibroblast-derived agents, or agents generated therefrom (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) results in modification of one or more types of cells present at the injury site. In specific embodiments, one or more agents are also included in the exposure and may or may not be provided exogenously, as in other cases where they are endogenous to the environment and / or cells and / or tissues.

[0018] In certain embodiments, the methods of the present disclosure occur ex vivo, such as in a culture, and ex vivo activate / inactivate fibroblasts or other cells in a treatment before application to a wound, for example. In certain embodiments, the methods are performed by human hands and do not encompass normal or random occurrences in the body. In certain embodiments, the methods of the present disclosure are unnatural. In certain embodiments, the concentrations of cells or cell-derived materials (fibroblasts; fibroblast-derived lysates; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) used in a method of exposing one type of cell to another type of cell do not occur in nature and do not occur randomly in nature.

[0019] The lack of natural exposure due to metabolic and immunodeficiency in diabetic patients may be one explanation for the non-healing or poor healing of their chronic wounds. In certain embodiments, the concentrations of one or more agents used in a method of exposing one or more types of cells with one or more agents do not occur in nature and do not occur randomly in nature. Any type of cell modification contained in this disclosure that occurs ex vivo or in vitro does not occur naturally in vivo in the same manner. In such embodiments, a tissue biopsy from a donor is used to isolate, characterize, activate, expand, and reintroduce one or more cells into the donor for the purpose of wound repair.

[0020] This disclosure encompasses therapeutic applications of cells including epithelial cells, neutrophils, macrophages, keratinocytes, vascular epithelial cells, fibroblasts (including myofibroblasts), and mixtures thereof. In at least some cases, fibroblasts or fibroblast-derived materials (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts) are modified before exposure to a wound site, such as through chemical, genetic, viral, physical, mechanical, or epigenetic activation and / or exposure to conditions not normally seen in the body. In other cases, neutrophils, macrophages, keratinocytes, vascular epithelial cells, myofibroblasts, or derivatives thereof are modified, such as by activation, before exposure to fibroblasts.

[0021] Embodiments of this disclosure provide means for utilizing fibroblasts as allogeneic, autologous (or heterogeneous or allogeneic) therapeutic cells through modification of culture conditions. In one embodiment of this disclosure, fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are extracted from a low immunogenic source (e.g., placental fibroblasts, omental fibroblasts, umbilical cord blood fibroblasts, etc.). In one embodiment of this disclosure, fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are extracted from the skin or any other area of ​​the body where a fibroblast-derived biological healing process is effective.

[0022] In one embodiment of the present disclosure, fibroblasts (fibroblasts; fibroblast-like cells; fibroblast-derived lysates; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are cultured in vitro to maintain the viability and proliferative capacity of the fibroblasts. The present disclosure provides modifications of known culture techniques to reduce recognition of fibroblasts by the recipient's immune system. In one embodiment, fibroblasts (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are cultured under heterogeneous conditions, such as a heterogeneous culture medium. In some embodiments, for example, the medium does not contain fetal calf serum. In certain embodiments, the Disclosure encompasses the substitution of fetal calf serum with one or more other agents, wherein such agents are, for example, those that promote a reduction in the immunogenicity of fibroblasts (e.g., human platelet-rich plasma, platelet lysates, umbilical cord blood serum, autologous serum, and / or one or more prescribed cytokines such as one or a combination of fibroblast growth factor, epidermal growth factor, leukemia suppressor, insulin-like growth factor, angiopoietin, and vascular endothelial growth factor).

[0023] In one embodiment of the present disclosure, an effective amount of fibroblasts (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) prepared by the methods encompassed by the present disclosure is administered to an individual for the treatment or prevention of one or more medical conditions. In certain embodiments, fibroblasts and / or fibroblast-derived materials are administered to improve wound repair, proliferation, cell differentiation, and tissue remodeling.

[0024] Embodiments of the present disclosure provide methods for co-administering universal donor fibroblasts or self-modified fibroblasts with one or more agents that stimulate and maintain the wound repair process. In particular embodiments of the present disclosure, methods are provided for co-administering universal donor fibroblasts and / or fibroblast-derived materials with one or more growth factors, chemokines, cytokines, and / or growth factors. In one embodiment of the present disclosure, universal donor fibroblasts derived from fibroblasts treated under conditions that reduce immunogenicity are utilized to stimulate fibroblast growth factors necessary for wound healing, such as EGF, FGF-2, TGF-β, PDGF, VEGF, IL-1, IL-6, or TNF-α, by introduced modified / unmodified fibroblasts or endogenous cells, thereby leading to tissue regeneration and wound healing

[10] .

[0025] Embodiments of this disclosure provide methods for reducing the immunogenicity of specific types of fibroblasts. Fibroblasts originate from a variety of tissues or organs, including but not limited to the skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, and foreskin, and can be obtained at biopsy (where appropriate) or autopsy. In some embodiments, the cells comprise fibroblasts, which may originate from a fetus, neonatal, adult, or a combination thereof.

[0026] Fibroblasts for use in any method of this disclosure may, in certain embodiments, be exposed to certain culture medium components.

[0027] The features and technical advantages of the present invention have been outlined in a rather general manner above. This is for the purpose of enabling a better understanding of the following detailed description of the present invention. Hereinafter, additional features and advantages of the present invention will be described, and these form the subject matter of the claims of the present invention. It should be understood by those skilled in the art that the disclosed concept and specific embodiments can be readily utilized as a basis for modifying or designing other structures for accomplishing the same objectives of the present invention. It should also be understood by those skilled in the art that such equivalent structures do not depart from the spirit and scope of the present invention as defined in the appended claims. The novel features believed to be characteristic of the present invention, both as to its organization and method of operation, together with further objectives and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. However, it should be explicitly understood that each drawing is provided for purposes of illustration and description only and is not intended as a definition of the limits of the present invention.

[0028] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, so it should be understood that the detailed description and specific examples are given for purposes of illustration only while showing specific embodiments of the present invention.

[0029] The following drawings form a part of this specification and are included to further illustrate specific aspects of the present disclosure. The present disclosure can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

[0030] [Figure 1A] FIG. 1A is a diagram showing the dietary components of a mouse model of human metabolic syndrome and obesity-induced type 2 diabetes.

[0031] [Figure 1B]Figure 1B shows the time course of achieving the desired baseline blood glucose level.

[0032] [Figure 2] Figure 2 shows an example of a wound treatment regimen.

[0033] [Figure 3] Figure 3 shows the daily progress of wound closure when control Matrigel is compared to locally provided fibroblasts (FB) (top image with control bar to the left of FB bar); when control Matrigel is compared to FB-derived exosomes (bottom left image with control bar to the left of FB bar); and when control Matrigel is compared to subcutaneously provided FB (bottom right image with control bar to the left of FB bar).

[0034] [Figure 4] Figure 4 shows the progression of wound closure comparing control Matrigel (top line) with subcutaneously donated fibroblasts (second line from the top), fibroblast exosomes (third line from the top), and locally donated fibroblasts (bottom line).

[0035] [Figure 5] Figure 5 shows typical images of wound healing and closure.

[0036] [Figure 6] Figure 6 shows an example of an experimental design for investigating the effects of freeze / thaw storage on fibroblast exosomes and fibroblast lysates.

[0037] [Figure 7] Figure 7 demonstrates the effects of frozen / thawed fibroblast exosomes and fibroblast lysates.

[0038] [Figure 8] Figure 8 shows the daily progression of wound closure: freeze / thaw implementation.

[0039] [Figure 9] Figure 9 shows wound closure using fibroblasts and fibroblast-derived exosomes.

[0040] [Figure 10] Figure 10 shows an example of a wound dressing product test on cell viability, particularly a test of 3M Nexcare® and ElaSkin® on mouse dermal fibroblasts.

[0041] [Figure 11] Figure 11 shows the effects on growth and cell size compared to untreated control cells.

[0042] [Figure 12] Figure 12 shows an example of a test design to investigate the effect of fibroblast spheroids on wound healing covered with 3M Nexcare®.

[0043] [Figure 13] Figure 13 shows fibroblast spheroids tested with 3M Nexcare® wound cover. In the left image, the Spheroids + 3M Nexcare® line is the bottom line. In the right image, the Spheroids + 3M Nexcare® bar is to the left of the 3M Nexcare® control bar.

[0044] [Figure 14] Figure 14 provides a representative image of wound healing using spheroids covered with 3M Nexcare®.

[0045] [Figure 15] Figure 15 shows an example of a study design to investigate the effects of fibroblast spheroids and lysates on wound healing in wild-type mice.

[0046] [Figure 16]Figure 16 shows the effects of spheroids and lysates using 3M Nexcare® on wild-type mice. In the upper left image, the spheroid + 3M Nexcare® line is the bottom line. In the lower left image, the spheroid + 3M Nexcare® bar is to the right of the 3M Nexcare® bar. In the upper right image, the FB Lysate + 3M Nexcare® line is below the 3M Nexcare® line. In the lower right image, the FB-derived lysate + 3M Nexcare® bar is to the right of the control 3M Nexcare® bar.

[0047] [Figure 17] Figure 17 shows images of fibroblast spheroids with 3M Nexcare® cover and 3M Nexcare® cover alone in wild-type mice.

[0048] [Figure 18] Figure 18 provides a representative image showing the effect of fibroblast lysates on wound closure in wild-type mice with 3M Nexcare® cover.

[0049] [Figure 19] Figure 19 provides an example of a study design to investigate the effects of a single treatment with mouse fibroblast spheroids and human fibroblast spheroids + 3M Nexcare® compared to Grafix®.

[0050] [Figure 20] Figure 20 shows a comparison of wound healing progression in mouse and human fibroblast spheroids compared with Grafix® in diabetic mice.

[0051] [Figure 21] Figure 21 provides representative images of wound closure using fibroblast spheroids compared to Grafix®.

[0052] [Figure 22] Figure 22 shows an example of fibroblast spheroid transplantation, migration, and proliferation on the wound surface. [Modes for carrying out the invention]

[0053] I. Definition example Throughout this application, the term "about" is used to indicate that the value includes inherent variations in the error of the measurement or quantification method. When used in combination with the term "comprising," the use of the words "a" or "an" can mean "one," but this is consistent with the meanings of "one or more," "at least one," and "one or more."

[0054] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that changes by the same amount as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a base quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In certain embodiments, when the term “about” or “approximately” precedes a numerical value, it indicates a value plus or minus a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term may mean within one order of magnitude of the value, e.g., within five times, and e.g., within two times. Unless otherwise specified, the term “about” means within an acceptable margin of error for a particular value.

[0055] As used herein, the terms “or” and “and / or” are used to describe multiple components, either in combination with each other or exclusively. For example, “x, y, and / or z” may refer to x alone, y alone, z alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is particularly intended that x, y, or z may be specifically excluded from embodiments.

[0056] Throughout this application, the term "about" is used in accordance with its plain and common sense in the field of cell and molecular biology to indicate that the value includes the standard deviation of errors in the apparatus or method employed to determine the value.

[0057] The terms “comprising” (and any form of “comprising” such as “comprises” and “comprises”), “having” (and any form of “having” such as “have” and “has”), “including” (and any form of “including” such as “includes” and “include”), or “containing” (and any form of “containing” such as “containing” and “contain”) mean inclusiveness or openness and do not exclude additional, unmentioned elements or methods or procedures.

[0058] The term "contains," which is synonymous with "includes," "encompasses," or "characterized by," is comprehensive or open-ended and does not exclude additional, unmentioned elements or method steps. The expression "consists of" excludes unspecified elements, steps, or components. The expression "essentially consists of" limits the scope of the described subject matter to the specified materials or steps and those that do not materially affect their basic and novel properties. Embodiments described in the context of the term "contains" are intended to also be carried out in the context of the terms "consists of" or "essentially consists of."

[0059] In accordance with long-standing patent law practice, the words “a” and “an” mean one or more when used in conjunction with the word “comprising” in this specification, including in the claims. Some embodiments of this disclosure may consist of, or be essentially, one or more elements, method steps, and / or methods of this disclosure. Any method or composition described herein may be carried out in relation to any other method or composition described herein, and different embodiments may be combined.

[0060] Throughout this specification, unless the context otherwise requires, the words “include,” “contain,” and “contain” will be understood to mean include the specified step or element or group of steps or elements, but not to mean exclude other steps or elements or groups of steps or elements. “Consists of” means to include and limit to what follows the phrase “consists of.” Thus, the expression “consists of” indicates that the listed elements are required or essential, and other elements may not be present. “Essentially consists of” means to include all elements listed after the phrase, and is limited to including other elements only if they do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the expression “essentially consists of” indicates that the listed elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0061] Throughout this specification, any reference to “one embodiment,” “a certain embodiment,” “a specific embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the occurrence of the aforementioned phrases in various places within this specification does not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0062] As used herein, the term “activated fibroblasts” refers to any type of fibroblast treated with one or more stimulants or agents capable of inducing one or more changes in fibroblasts (metabolism, immunity, epigenetics, growth factor secretion, surface marker expression, and microvesicle formation and excretion). Examples of agents include at least ADP, CXCL4, PDGF, TGFβ, IL-1, TNFα, ROS, NO, AMP, IL-17, IL22, dsRNA 1.25D3, IL-8, CXCL1-8, Activin A, β-defensins, catheteridin, E-selectin, P-selectin, KGF, VEGF, IGF, IL-10, IL-4, and IL-13.

[0063] As used herein, the term “activated immune cells” means immune cells treated with one or more stimuli or agents capable of inducing one or more intracellular changes, including metabolic, immunological, epigenetic, growth factor secretion, surface marker expression, and microvesicle generation and excretion. Examples of agents include at least ADP, CXCL4, PDGF, TGFβ, IL-1, TNFα, ROS, NO, AMP, IL-17, IL22, dsRNA 1.25D3, IL-8, CXCL1-8, Activin A, β-defensins, cathelicidin, E-selectin, P-selectin, KGF, VEGF, IGF, IL-10, IL-4, and IL-13.

[0064] As used herein, the terms “administered” or “to administer” refer to any method by which a composition is provided to an individual in such a way that the composition has the intended effect on a patient. For example, one method of administration is by indirect mechanisms using medical devices such as catheters, applicator guns, syringes, cells or cell products loaded into gel matrices, synthetic polymers or biopolymers, and 3D matrices containing one or more cell types, cell-derived products and / or growth factors and / or antibiotics. A second exemplary method of administration is by direct mechanisms such as topical tissue administration, oral ingestion, transdermal patches, topical administration, inhalation, and suppositories.

[0065] As used herein, “same species” means tissue or cells from one or more individuals of the same species, but from another body that is immunologically incompatible or capable of being immunologically incompatible in the natural environment.

[0066] As used herein, “autologous” refers to tissue or cells derived from or transplanted from the body of the same individual (i.e., autologous blood donation; autologous bone marrow transplant).

[0067] As used herein, “agent” means nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof, including whole cell lysates.

[0068] As used herein, “heterogeneous” refers to tissue or cells from a species different from that of the patient.

[0069] Cell culture refers to an artificial in vitro system containing viable cells, whether in the quiescent, senescent, or actively dividing phases. In cell culture, cells are grown and maintained at an appropriate temperature, typically 37°C, and in an atmosphere typically containing varying concentrations of oxygen and CO2. However, culture conditions can vary significantly depending on the cell type, and changing the conditions for a particular cell type can result in different phenotypes. The most commonly variable factors in a culture system are the culture medium and the oxygen concentration during culture. Growth media vary in the concentration of nutrients, growth factors, and other components. Growth factors used to supplement the medium are often derived from animal blood, such as calf serum.

[0070] As used herein, the term “individual” refers to a person or animal which may or may not be admitted to a medical facility and which may be treated as an outpatient of a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may constitute any age of human or non-human animal and therefore include both adults and adolescents (i.e., children) and infants. The term “individual” is not intended to imply a need for medical treatment, and therefore an individual may participate in an experiment voluntarily or involuntarily, whether clinical or in support of basic scientific research. The terms “subject” or “individual” may be used interchangeably and refer to any living organism or animal subject to a method or substance, such as mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0071] Throughout this specification, any reference to “one embodiment,” “a certain embodiment,” “a specific embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Therefore, the occurrence of the aforementioned phrases in various places within this specification does not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0072] When referring to the occurrence of any symptom in an untreated subject compared to a treated subject, the terms “reduction,” “inhibition,” “attenuation,” “suppression,” “decrease,” “prevention,” and their grammatical equivalents (including “lower,” “smaller,” etc.) mean that the amount and / or magnitude of the symptom in the treated subject is lower than in the untreated subject by any amount recognized as clinically relevant by any medically trained person. In one embodiment, the amount and / or magnitude of the symptom in the treated subject is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the amount and / or magnitude of the symptom in the untreated subject.

[0073] As used herein, the term “transplant” refers to the process of taking living tissue or cells and transplanting them to another part of the body or to another body.

[0074] As used herein, the term “therapeutably effective amount” means the amount of composition provided to an individual such that a wound is improved in terms of severity and / or size (including diameter or area).

[0075] "Treatment," "treatment," or "to treat" means a method of reducing the effects of a disease or condition. Treatment may also refer to a method of reducing the disease or condition itself, as well as symptoms. Treatment may be any reduction from a pre-treatment level and may be, but not limited to, the complete excision of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed method, "treatment" may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disease progression, including a reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered a treatment if, in the same or a control subject, there is a detectable reduction in the immunogenicity of cells compared to a pre-treatment level. Thus, the reduction may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, compared to the native or control level. It is understood and intended hereby understood that "treatment" does not necessarily mean a cure for the disease or condition, but rather an improvement in the prognosis for the disease or condition. In specific embodiments, treatment may mean reducing the severity or degree of at least one symptom, or alternatively or additionally, delaying the onset of at least one symptom.

[0076] In specific embodiments, the application of externally applied fibroblasts and / or fibroblast-derived materials (including fibroblasts; fibroblast-like cells; fibroblast-derived lysates; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts) to a wound can initiate, maintain, and accelerate the wound healing process in all types of injuries, including diabetic ulcers, non-healing wounds, surgical incisions, trauma, abrasions, skin disorders, cuts, and burns. As used herein, the term “wound” encompasses, for example, injury to living tissue caused by a cut, blow, or other impact, typically resulting in a cut or tear of the skin. In specific embodiments, all of the above conditions are wounds that require healing.

[0077] As used herein, the term “fibroblast-derived material” means fibroblast fragments, conditioning culture media, exosomes secreted from fibroblasts, and / or fibroblast lysates; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts.

[0078] The term "fibroblast-like cells" refers to cells that share certain characteristic features with fibroblasts but exhibit similar surface markers, structural features, expression patterns, and epigenetic profiles to closely related cells in specific locations. Examples include myofibroblasts, perineurial sheath cells, lymphoid organ dendritic cells, and pilar fibroblasts.

[0079] Embodiments of this disclosure encompass the use of cells and / or cell products for wound healing, including initiating and / or maintaining wound healing. In some embodiments, cells and / or cell products are exposed to one or more compositions and / or environments before or during use for wound healing to enhance their use in wound healing. In specific embodiments, cells and / or cell products are exposed in an in vitro setting to generate activated fibroblasts, activated immune cells, fibroblast-derived materials from activated keratinocytes, activated angioepithelial cells, activated myofibroblasts or their fibroblast derivatives, including for introduction into, on, and / or adjacent to a wound, in order to initiate or maintain wound healing. In some embodiments, the cells used are not activated.

[0080] Embodiments of the present disclosure involve introducing fibroblasts or fibroblast-derived materials (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts), fibroblasts activated with one or more agents, and / or fibroblasts (including myofibroblasts or dermal fibroblasts) plus one or more immune cells, keratinocytes, and / or vascular epithelial cells, and / or derivatives thereof, into the middle, upper, and adjacent areas of a wound for the purpose of initiating or maintaining wound healing.

[0081] The methods of this disclosure can be used for any abrasion (a wound caused by friction when the body rubs against a rough surface), delamination (characterized by a flap), incision (a cut with a clean edge), laceration (a cut with a jagged edge), or puncture (a wound caused by something passing through or piercing the skin). The wound may or may not be infected with a pathogen, and if infected, one or more antibiotics may be used on the wound and / or systemically on the individual. In such cases, the antibiotics and the compositions incorporated herein may or may not be administered simultaneously, and may or may not be in the same composition. The wound may be a Class 1, 2, 3, or 4 wound as classified by the U.S. Centers for Disease Control and Prevention.

[0082] In various embodiments, the introduction of fibroblasts and / or fibroblast-derived materials (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts), fibroblasts activated with one or more agents, and / or fibroblasts and one or more types of immune cells (including their derivatives, which may be activated) on and / or adjacent to a wound brings about partial or complete healing, for example, (1) tissue differentiation; (2) cell recruitment and differentiation of stem cells at the wound site; (3) expansion of keratinocytes, vascular epithelial cells, myofibroblasts, and / or dermal fibroblasts that are already locally present; and / or (4) induction to assist the healing of other cells or biological processes, all of which are carried out with the aim of initiating or maintaining wound healing. In certain embodiments, wound healing by any mechanism occurs at a faster rate than wound healing in the absence of the disclosed methods or compositions.

[0083] In certain embodiments, fibroblasts (including fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing fibroblast exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components) or other cells incorporated herein become activated after exposure to one or more conditions and / or agents. Certain agents that can modify fibroblasts include one or more nucleic acids, cytokines, chemokines, growth factors, and / or exosomes before and / or during their use. Fibroblasts may be activated, such as by modifying nucleic acids to have activated or manipulated surface markers, and / or by having the expression or excretion of one or more chemokines, one or more cytokines, exosomes, and / or one or more growth factors. Cells may be activated by one or more chemical agents, RNA, microRNA, RNAi, DNA, viral nucleic acids, and / or exosomes. In specific embodiments, cytokines are selected from the group consisting of IFN-γ, TNF-α, interleukin (IL)-1, IL-6, IL-7, IL-8, IL-12, IL-15, IL-17, IL-33, and combinations thereof. In specific embodiments, growth factors are selected from the group consisting of FGF-1, VEGF, and combinations thereof. In specific embodiments, cells are activated after exposure to human platelet-rich plasma, platelet lysates, umbilical cord blood serum, autologous serum, human serum, serum substitutes, or combinations thereof. In specific embodiments, cells are activated after exposure to hypoxia. Hypoxia may be, for example, 0.1% to 10%, 0.1% to 5%, 0.1% to 2.5%, or 0.1% to 1% oxygen. In certain embodiments, hypoxia occurs for a period of at least about 30 minutes to about 3 days, or longer, but in some cases it may be less than 30 minutes or longer than 3 days. Fibroblasts may be exposed to one or more growth factors before and / or during exposure to hypoxia, carbon monoxide, or a combination thereof.

[0084] In some embodiments, fibroblasts or fibroblast-derived materials (fibroblasts; fibroblast-like cells; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are used with one or more adjuvants to activate the immune system and initiate or maintain the wound healing process. These adjuvants may be chemical, mechanical, viral, genetically modified, or bacterial product-based. Examples include water-in-oil emulsions containing at least aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate monophosphoryl lipid A, Qs-21, cytosine phosphoguanine, saponins, and / or squalene.

[0085] In certain embodiments, encapsulated RNA, microRNA, or RNAi, and / or fibroblast-derived exosomes or other microvesicles, any other cells or cell fragments derived from fibroblasts are utilized to enable fibroblasts to initiate and maintain the local wound healing process.

[0086] Embodiments of the present disclosure include the use of fibroblasts or fibroblast-derived materials (including fibroblasts; fibroblast-like cells; and extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) in angiogenesis, repair, and / or regeneration at wound sites and surrounding tissues.

[0087] In certain embodiments, a therapeutically effective amount of spheroids may be applied to a wound. This amount may be determined by the number of cells in the spheroids and / or the number of spheroids applied. For example, in the case of a wound with a diameter of 8 mm, approximately 100 spheroids, corresponding to approximately 3 million cells, may be applied to the wound. In certain embodiments, at least approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 750, or 1000 or more spheroids may be applied to a wound. Each spheroid may contain at least approximately 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, or more cells.

[0088] In certain embodiments, therapeutically effective amounts of lysates, exosomes, etc., may be obtained from a specific amount of fibroblasts cultured in a culture medium. In certain embodiments, the amount of cultured fibroblasts includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 × 1 million or more cells. Specifically, the number of cultured fibroblasts to produce lysates, exosomes, etc., is determined by the size of the wound being treated. In one embodiment, a wound of about 8 mm is provided with lysates or exosomes from 3 million fibroblasts. In certain embodiments, the amount of material administered will depend on the size of the wound. In certain embodiments, the amount of material administered is 1 mm 2 This ranges from 30,000 to 100,000 cells, or the amount of material obtained from that number of cells.

[0089] In some embodiments, one or more compositions are present in, on, around, or in combination on a substrate, or delivered onto a substrate that constitutes a composition. In other embodiments, the composition is applied to the substrate at the required site. In some embodiments, one or more compositions may or may not be used in the same wound or burn, and the same type of substrate may or may not be employed.

[0090] In certain embodiments of the composition, one or more base components consist of or contain synthetic polymers and / or biopolymers, and in certain cases the biopolymer is a natural polymer. The synthetic polymer and / or biopolymer may be of any type, as long as it is suitable for use on and / or in a body. In embodiments where the base of the composition consists of biopolymers such as keratin, fibrin, collagen, cellulose, alginic acid, chitosan, cyanofuran, hyaluronic acid, and / or hydrogels. In embodiments where the polymer comprises one or more synthetic polymers, the synthetic polymers are poly(ethylene glycol) (PEG) / poly(ethylene oxide) (PEO), poly(vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA),

[15] , poly(hydroxyethyl methacrylate) (PHEMA), polyurethane (PU), poly(α-ester) [e.g., poly(lactic acid-coglycolic acid) (PLGA), polyglycolic acid (PGA), polylactide (PLA), and / or poly(ε-caprolactone) (PCL). In embodiments where the polymer consists of one or more synthetic inorganic polymers such as PDMS, HPE, natural minerals, synthetic silicates, phosphates, zeolites, clays, cesoporous silica, and poly(P)).

[0091] The constituent parameters of the substrate, including at least size, material, thickness, the presence of any one or more compartments, the presence of any one or more openings, and shape, may or may not be tailored to the intended wound / burn. In some embodiments, the substrate is modified with constituent parameters(s) for the intended use of the composition, while in other cases, the substrate is used off-the-shelf. If the substrate is modified for a specific application, the composition may or may not already be present on the substrate. If the substrate is not modified initially and is used off-the-shelf, the composition may or may not already be present on the substrate.

[0092] The shape of the outer frame of the base material may be random, or it may be a specific shape such as a square, rectangle, triangle, circle, trapezoid, rhombus, or crescent. Therefore, the base material may be a specific geometric formation. The width of the base material applied to a site may depend on the width of the wound. The base material may be reduced to a size that fits the wound before or after the base material is placed on or around the wound. The size and shape of the base material can be adjusted to the size of the wound.

[0093] In some embodiments, the substrate may be a scaffold. In specific embodiments, the scaffold facilitates the placement of single-cell or spheroidal cells within the scaffold and allows for cell migration and / or proliferation from the location where the cells are placed, for example, in the dermis. In some embodiments, the scaffold allows for tissue growth to mimic biological processes or structures for tissue engineering where replacement is needed. In certain embodiments, the scaffold may be fibrous, porous, or hydrogel.

[0094] In specific embodiments, the substrate may be a tray, cup, mesh, etc. The substrate may be composed of any kind of biopolymer, organic or inorganic polymer fabric.

[0095] In specific embodiments, the substrate comprises one or more openings or perforations. The openings or perforations in the substrate may be in a random pattern or an ordered pattern. The size of the openings may be configured to be large enough to allow efficient cell migration and to allow one or more spheroids to be fixed to a site.

[0096] In certain embodiments, single-cell or spheroidal fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, apoptotic bodies from fibroblasts, and / or fibroblast-related products, or any combination thereof, are introduced on any type of substrate, such as a scaffold, tray, geometric formation, biological cloth, mesh, or polymer cloth made of a synthetic polymer or biopolymer. The step(s) of applying the biological component(s) to the substrate component(s) may be any suitable embodiment, whether before or during placement in a wound or burn.

[0097] In certain embodiments, devices such as dispensing devices are used to introduce single-cell or spheroidal fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies and / or fibroblast-related products from fibroblasts onto scaffolds, trays, geometric formations, biological fabrics, meshes, or polymer fabrics of synthetic polymers or biopolymers. Any dispensing device referred to herein may be considered an applicator.

[0098] In certain embodiments, a device such as a dispensing device designed to have one or more grooves is used to introduce single-cell or spheroidal fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies from fibroblasts into a scaffold, tray, geometric formation, biological fabric, mesh, or polymer fabric of a synthetic polymer or biopolymer.

[0099] In certain embodiments, a device such as a dispensing device is used to introduce single-cell or spheroidal fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies and / or fibroblast-related products from fibroblasts into a scaffold, tray, geometric formation, biological fabric, mesh, or polymer fabric of a synthetic polymer or biopolymer. In certain embodiments, the device is designed to have an ordered pattern of holes to allow for the placement of spheroids, or a random sequence of holes to allow for the placement of random spheroids.

[0100] In some embodiments, a spray or misting device is used to introduce single-cell or spheroidal fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies and / or fibroblast-related products from fibroblasts onto scaffolds, trays, geometric formations, biological cloths, meshes, or polymer cloths of synthetic polymers or biopolymers. In certain embodiments, the spray or misting device is used to ensure uniform distribution of the biological material onto the substrate.

[0101] In certain embodiments, a 3D printer is used to introduce single-cell or spheroid fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies and / or fibroblast-related products from fibroblasts onto a substrate made of synthetic polymers and / or biopolymers, such as a scaffold, tray, geometric formation, biological cloth, mesh, or polymer cloth.

[0102] In certain embodiments, there is a culture of fibroblast spheroids within the pores of a porous synthetic polymer or biopolymer, where the openings are spaced apart in such a way that they accommodate the proliferation of spheroids containing a specific size and number of fibroblasts onto the wound surface.

[0103] In some embodiments, fibroblast spheroids of a specific size are dispensed into openings of a biomolecular substrate so that only one spheroid is positioned in each opening, allowing for the proliferation of fibroblasts to the wound surface from the opposite side of the opening.

[0104] In certain embodiments, a high-resolution camera is used to generate images for introducing single-cell or spheroid fibroblasts, exosomes from fibroblasts, lysates from fibroblasts, or apoptotic bodies and / or fibroblast-related products from fibroblasts into a 3D printer, for the purpose of matching the boundaries of the wound to be covered. [Examples]

[0105] The following embodiments are included to illustrate specific embodiments of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that have been found to work well in the implementation of the present invention and can therefore be considered to constitute specific aspects for implementation. However, those skilled in the art should understand that many modifications can be made in light of the present disclosure to the specific embodiments disclosed, and that similar or analogous results can still be obtained without departing from the spirit and scope of the embodiments of the present disclosure.

[0106] Example 1 Therapeutic use of fibroblasts for wound healing Introduction

[0107] Skin wound repair is essential for the survival of organisms, and impaired skin repair, leading to wounds that do not heal, is a major health problem worldwide. Wound healing research is complex, mainly due to the multifaceted nature of the wound environment and the complexity of the healing process, which integrates various cellular and repair stages such as inflammation, proliferation, re-epithelialization, and remodeling. Delayed wound healing of the feet is a serious complication caused by hyperglycemia in patients with type 2 diabetes (DM), and these wounds can develop into foot ulcers. Studies have shown that wounds in diabetic patients heal at less than 50% the rate of wounds in non-diabetic patients 30 days after the injury compared to wounds in non-diabetic patients.

[0108] Several factors contribute to delayed wound healing. These include impaired fibroblast proliferation and migration, hyperglycemia, and impaired keratinocyte proliferation and migration. Another major cause of impaired wound healing is an optimal immune response, increased reactive oxygen species, regulated angiogenesis, and persistent bacterial growth at the wound site. Skin fibroblasts are involved in all stages of wound healing, including hemostasis, inflammation, proliferation, and remodeling. Furthermore, elucidating the mechanisms of chronic wounds remains a major challenge due to the lack of suitable genetic mouse models. This disclosure demonstrates the effects of fibroblasts in diabetes-induced ulcers and explains the promotion of wound healing by fibroblasts and fibroblast-derived materials.

[0109] Methods and materials

[0110] mouse

[0111] A polygenetic model (leptin gene, leptin receptor (LEPR), Ay gene mutations) was used in the study. 10-12 week old NONcNZO10 / LtJ mice from Jacksons Laboratory were used. The mice were individually placed in ventilated cages and housed in a 12-hour light-dark cycle. Once the desired baseline blood glucose level of 300 mg / dL was reached (Figure 1B), the mice were placed on a high-fat diet (F3282 pellets) containing 36% fat (Figure 1A).

[0112] Perform cell culture.

[0113] Mouse embryonic fibroblasts were obtained from the American Type Culture Collection (ATCC®), cultured in DMEM High Glucose, and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, respectively. The cells were cultured at 37°C and 5% CO2.

[0114] Isolation of fibroblast-derived exosomes

[0115] Preparation of Conditional Culture Medium

[0116] Fibroblasts were plated and cultured for 24 hours in DMEM medium containing 10% FBS. They were then washed three times with PBS and finally cultured for 2 hours in 3 ml of serum-free DMEM medium. The culture medium was collected and filtered through a 0.22 μm filter to remove cell residue.

[0117] Isolation and characterization of exosomes released from fibroblasts

[0118] Exosomes were isolated from cultured fibroblasts using the Total Exosome Isolation Kit. Briefly, 0.5 ml of total exosome isolation reagent was added to 1 ml of filtered conditional medium and thoroughly mixed by inversion. After incubation overnight at 4°C, the cells were centrifuged at 12,000 × g at 4°C for 70 minutes, and the supernatant was completely aspirated and removed. The exosome pellet was resuspended in a very small amount of DMEM medium without dilution before use.

[0119] In vivo processing

[0120] Cell preparation technology

[0121] Cells are cultured in a T225 flask at 37°C. When the cells reach 70% confluence, they are trypsinized with 0.5% trypsin and centrifuged at 400g for 5 minutes. Discard the supernatant and resuspend the pellet in PBS. The cells are then incubated in PBS at 1x10⁻¹⁶. 6 Dispense it.

[0122] Full-thickness wound model

[0123] Mice were anesthetized using isoflurane inhalation. Mice were shaved and sterilized with betadine and alcohol. On day 0, four full-thickness wounds were made on the back on both sides of the midline at shoulder level using a sterile 8 mm punch biopsy. The skin was lifted using serrated forceps, and full-thickness wounds were made through the subcutaneous tissue using iris scissors.

[0124] Wound treatment

[0125] Mice were anesthetized using isoflurane inhalation. Mice were grouped into four treatment groups: Matrigel, topical fibroblasts (FB(Top)), subcutaneously delivered fibroblasts (FB(SC)), and fibroblast-derived exosomes (FB-derived exosome). Each therapeutic compound was either gently applied to the wound or administered subcutaneously around the wound. All mice received a total of five treatments (days 1, 3, 5, 7, and 9) (Figure 2).

[0126] Wound measurement

[0127] Wounds were measured daily. Mice were anesthetized using 5% isoflurane inhalation. Wound diameter and area were measured using the eKare® insight advanced wound imaging device, which is also approved for clinical use. After measurement, a clean occlusive dressing was reapplied, and the animals were kept warm until full recovery. Wound closure of four wounds was averaged per mouse.

[0128] histology

[0129] Skin fragments containing the wound were excised and used for further analysis. For H&E staining, tissue samples were collected on day 20, fixed with 10% NBF, and embedded in paraffin. H&E staining was performed using the ST Infinity H&E staining system, and Sirius Red staining was performed with 0.1% picrosirius red. H&E-stained slides were imaged.

[0130] Immunolabeling of tissues

[0131] For immunofluorescence with CD31, tissues were fixed in formalin and embedded in paraffin. 5-micrometer sections were rehydrated, and antigen recovery was performed in 10 mM citrate buffer (pH 6.0) for 15 minutes to 1 hour. After blocking with 1% BSA at room temperature for 1 hour, sections were incubated overnight with primary antibody (CD31, Thermo Fisher) at 4°C, and then incubated with secondary antibody (Alexa Fluor 568, goat, anti-rat, Thermo-Fisher) at room temperature for 1 hour.

[0132] For paraffin-embedded samples, the tissue was fixed with 4% buffered paraformaldehyde (PFA) for 20 minutes and then permeabilized with 0.3% Triton-X for 30 minutes. After blocking with 1% BSA (at room temperature for 30 minutes), the sections were incubated overnight at 4°C with the primary antibody (Cytokeratin 5, Thermo Fisher), followed by incubation with the secondary antibody (Alexa Fluor 546, goat anti-rabbit) at room temperature for 1 hour, and then with nuclear staining (DAPI) for 30 minutes.

[0133] Quantitative real-time PCR

[0134] RNA was isolated from snap-frozen tissue and extracted with Trizol reagent according to the manufacturer's instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit. Real-time quantitative PCR was performed using the SYBR Green Real-Time PCR Master Mix.

[0135] Enzyme-linked immunosorbent assay

[0136] serum recovery

[0137] After allowing the blood to coagulate at room temperature for 2 hours, the serum was separated by centrifugation at 2000g for 15 minutes, and the assay was started immediately.

[0138] Tissue biopsy samples. Biopsy samples from healed wounds were collected at several points during the experiment, flash-frozen, and stored. Lysates from these tissues were used in cytokine and growth factor assays.

[0139] Mouse cytokine array analysis:

[0140] Mouse cytokine antibody arrays were prepared using the Abcam Mouse cytokine antibody array (120 targets - ab197461) kit, following the manufacturer's instructions.

[0141] List of cytokines detected by array analysis;

[0142] Tumor necrosis factor (TNF-α).

[0143] Interleukin-6 (IL-6).

[0144] IL-1β.

[0145] Interferon-γ (IFN-γ).

[0146] IL10.

[0147] Epidermal growth factor (EGF).

[0148] Vascular endothelial growth factor (VEGF).

[0149] result

[0150] Localized fibroblasts promote wound healing in diabetic patients.

[0151] To demonstrate that locally administered fibroblasts promote wound healing, 10-12 week old NONcNZO10 / LtJ mice were fed a high-fat diet of 36% fat after their baseline blood glucose levels reached the desired 300 mg / dL. At 300 mg / dL, the mice were randomly assigned to one of four treatment groups: Matrigel, topical fibroblasts (FB(Top)), subcutaneous fibroblasts (FB(SC)), and fibroblast-derived exosomes (FB-derived exosomes). Four 8 mm wounds were created on the backs of each mouse, and 50 μL of each therapeutic agent was administered mixed with Matrigel. The diameter of these wounds was measured every other day until healing, and the mice were euthanized for further analysis. The efficacy of the treatment was evaluated by plotting the wound diameter and area using diameter and area data generated by the eKare® insight advanced wound imaging device, which is also approved for clinical use.

[0152] Experiments and analysis of the collected data showed that mice administered FB (topical) exhibited accelerated wound healing compared to a control group administered only Matrigel (Figure 3). Furthermore, in measurements of wound area, mice administered FB (topical), followed by FB-derived exosomes, and then FB(SC) showed significantly improved wound size compared to the control group. These findings confirm that FB (topical) promotes wound healing in diabetic wounds, followed by fibroblast-derived exosomes.

[0153] Significance of a Specific Embodiment

[0154] This study demonstrated that topically administered fibroblasts significantly increased wound healing rates compared to controls. Furthermore, mice administered fibroblast-derived exosomes showed substantial and significant improvements. These results suggest that directly applying fibroblasts or fibroblast-derived materials and allowing them to migrate and proliferate in the dermis accelerates the wound healing rate of diabetic wounds. The passive accumulation of fibroblasts at the wound site, and the established role of fibroblasts in immunomodulation, trigger an immune response that modulates angiogenesis, thereby accelerating wound healing. Additionally, fibroblasts and fibroblast-derived materials enhance healing rates, and fibroblast immunomodulation can prevent bacterial colonization, a known factor in delayed healing of diabetic wounds. These findings represent an important verification step for enhancing the therapeutic effects of fibroblast therapy.

[0155] summary

[0156] As a result of continuing trials of several therapeutic methods for delivering fibroblasts and fibroblast-derived materials to wound sites, it was found that topically applied fibroblasts and fibroblast-derived exosomes significantly improved the healing rate. This finding addresses a major problem in wound healing in diabetic patients and is useful for treating this chronic disease associated with diabetes.

[0157] Example 2 Wound care In one in vivo study, the inventors tested the administration of single-cell fibroblasts to wounds in a diabetic mouse model. One million single-cell fibroblasts were administered topically or subcutaneously around the wound margin. One million fibroblast exosomes derived from fibroblast culture medium, used to proliferate the single-cell fibroblasts, were also tested for topical administration. The topical cells and exosomes were mixed with Matrigel® immediately before application to ensure that the cells and exosomes remained on the wound. Only Matrigel® was used as a control. All control and test samples were administered every other day after measuring the diameter and area of ​​the wound. Wound healing was determined when the scab peeled off and epithelialization was confirmed (Figures 2-7).

[0158] The results of this study showed that when single-cell fibroblasts were administered locally and subcutaneously, wound healing was significantly faster than in the control group, similar to fibroblast-derived exosomes. Of the three test materials, single-cell fibroblasts and fibroblast-derived exosomes showed significantly better results than subcutaneously injected fibroblasts.

[0159] In another study, the objective of an in vivo experiment was to test the efficacy of unicellular fibroblasts and fibroblast-derived exosomes that had been pre-frozen (e.g., in liquid nitrogen) and thawed immediately before use. One million unicellular fibroblasts were administered topically. One million fibroblast exosomes derived from fibroblast medium, used to proliferate the unicellular fibroblasts, were also tested for topical administration. The topically administered cells and exosomes were mixed with Matrigel® immediately before application to ensure retention of the cells and exosomes in the wound. Only Matrigel® was used as a control. All control and test samples were administered every other day after measuring the diameter and area of ​​the wound. Wound size was measured every other day using the eKare® device, which is clinically approved for monitoring chronic wound healing. The eKare® device can also image the wound. Wounds were considered healed when the scab was released from the wound and epithelialization was confirmed (see Figures 6–9).

[0160] The results of this study showed that topical administration of frozen / thawed single-cell fibroblasts significantly accelerated wound healing compared to the control group, as did exosomes derived from frozen / thawed fibroblasts. This study demonstrated that freezing / thawing cells or exosomes does not affect the efficacy of the material for wound healing.

[0161] Another study aimed to test alternative and simple methods for covering wounds after topical administration of fibroblasts or fibroblast-derived exosomes to the wound. Since Matrigel is an animal product and is not currently approved for human use, other products approved for human wound dressing were used. In this study, the inventors tested 3M Nexcare® and ElaSkin® by applying the wound dressing to the surface of the culture site, inoculating with fibroblasts, and monitoring cell growth in terms of cell number and cell size using Agilent's IncuCyte® device.

[0162] The results of this study showed that 3M Nexcare® did not affect cell growth efficiency or size, while ElaSkin® significantly affected not only cell growth but also cell size, thus negatively impacting cell growth. Based on these findings, subsequent studies used 3M Nexcare® to coat wounds with fibroblasts and fibroblast-derived exosomes (Figures 10-11).

[0163] In additional studies, the objective of in vivo experiments was to test the administration of fibroblasts proliferated as spheroid organoids to wounds generated in a diabetic mouse model. 100 fibroblast spheroids, each containing approximately 30,000 fibroblasts (±15%), were administered topically to the wounds. Subsequently, 30-50 μl of 3M Nexcare® was applied to cover the wounds. 3M Nexcare® alone was used as a control. Spheroid and control samples were applied only once to test the one-time application of spheroid fibroblasts. Wound size was measured every other day using the eKare® device, which is clinically approved for monitoring chronic wound healing. The eKare® also captured images of the wounds. Wounds were considered healed when the scab was released from the wound and epithelialization was confirmed (Figures 12-14).

[0164] The results of this experiment showed that administered globular fibroblasts healed wounds significantly faster than the control group. Not only did wounds heal approximately 3 days faster than the control group, but inflammation in wounds treated with fibroblasts was significantly less than in the control group.

[0165] In one study, the objective of an in vivo experiment was to test the administration of fibroblasts proliferated as spheroid organoids, and unicellular fibroblast-derived lysates, to wounds in a wild-type non-diabetic mouse model. 100 fibroblast spheroids, each containing approximately 30,000 fibroblasts (±15%), were administered to the wound. Lysates obtained by mechanically lysing 1 million unicellular fibroblasts were also administered to the wounds for testing. The wounds were then covered with 30-50 μl of 3M Nexcare®. As a control, the inventors tested 3M Nexcare® alone. Spheroid, fibroblast lysate, and control samples were applied only once to test single-application efficacy. Wound size was measured every other day using an eKare® device, which is clinically approved for monitoring chronic wound healing. The eKare® device also captured images of the wounds. A wound was considered healed when the scab was released from the wound and epithelialization was confirmed (Figures 17-20).

[0166] The results of this study showed that globular fibroblasts and fibroblast-derived lysates significantly accelerated wound healing compared to controls, even in non-diabetic wounds. When comparing fibroblast spheroids with lysates, fibroblast spheroids healed wounds significantly faster than lysates.

[0167] In another study, the objective of an in vivo study was to test the administration of both mouse dermal fibroblasts and human dermal fibroblasts, proliferated as spheroid organoids, to wounds generated in a diabetic mouse model. Furthermore, the inventors wanted to test a commercially available product called Grafix®, which is FDA approved for use in the treatment of chronic wounds. 100 fibroblast spheroids, each containing approximately 30,000 fibroblasts (plus or minus 15%), were administered topically to the wounds. The inventors also tested the application of Grafix® to the wounds according to the application directions described in the Grafix® publication. Subsequently, 30-50 μl of 3M Nexcare® was applied and the wounds were covered. As a control, the inventors tested 3M Nexcare® alone. Human and mouse fibroblast spheroids, Grafix®, and control samples were applied only once to test the one-time application of spheroid fibroblasts and Grafix®. Wound size was measured every other day using the eKare® device, which is clinically approved for monitoring chronic wound healing. The eKare® device also took images of the wounds. Wounds were considered healed when the scab was released from the wound and epithelialization was confirmed (Figures 21-24).

[0168] This study demonstrated that human dermal fibroblast spheroids healed wounds significantly faster than controls, mouse dermal fibroblast spheroids, and Grafix®. Wounds healed on day 5 with human dermal fibroblast spheroids, on day 7 with mouse dermal fibroblast spheroids, and on day 8 with Grafix®. Furthermore, human dermal fibroblast spheroids significantly suppressed inflammation compared to other test materials, including preventing an increase in wound size after administration. In fact, wound size decreased immediately after administration. Comparing human dermal fibroblast spheroids with mouse dermal fibroblast spheroids, fibroblast spheroids showed remarkably faster wound healing.

[0169] All methods disclosed and claimed herein can be manufactured and performed without excessive experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps or the order of steps herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein, while the same or similar results are achieved. All such similar substitutions and modifications, which are apparent to those skilled in the art, are considered to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0170] References The following references are incorporated herein by reference insofar as they provide exemplary procedures or other details that supplement those described herein. 1.Chuong, CM, et al., What is the 'true' function of skin? Exp Dermatol, 2002. 11(2): p. 159-87.

[0171] 2.Bainbridge, P., Wound healing and the role of fibroblasts. J Wound Care, 2013. 22(8): p. 407-8, 410-12.

[0172] 3.Tracy, LE, RA Minasian, and EJ Caterson, Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound. Adv Wound Care (New Rochelle), 2016. 5(3): p. 119-136.

[0173] 4.Darby, I.A. and T.D. Hewitson, Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol, 2007. 257: p. 143-79.

[0174] 5.Shah, J.M., et al., Cellular events and biomarkers of wound healing. Indian J Plast Surg, 2012. 45(2): p. 220-8.

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Claims

1. A composition for use in a method of treating one or more wounds in an individual, the composition comprising fibroblasts and / or fibroblast-derived products, The method includes the step of administering a therapeutically effective amount of the composition to an individual. composition

2. The composition according to claim 1, wherein the fibroblast-derived product comprises extracellular vesicles, exosomes, microvesicles, apoptotic bodies, and / or fragments or biological components of fibroblasts.

3. The composition according to claim 1 or 2, further comprising administering to an individual one or more therapeutically effective amounts of immune cells, keratinocytes, vascular epithelial cells and / or substances derived therefrom.

4. The composition according to any one of claims 1 to 3, wherein the fibroblasts are myofibroblasts.

5. The composition according to any one of claims 1 to 4, wherein the fibroblasts are self, homogeneous, or heterogeneous with respect to the individual.

6. The composition according to any one of claims 1 to 5, wherein the cells are activated, manipulated, and / or catalyzed.

7. The composition according to claim 4, wherein cells are activated, manipulated, and / or catalyzed by exposure to mechanical means and / or the use of biological or genetic agents.

8. The composition according to claim 7, wherein the mechanical means includes light, laser, pressure, and / or stress.

9. The composition according to any one of claims 1 to 8, wherein cells are activated by exposure to cytokines, chemokines, growth factors, chemicals, RNA, microRNA, RNAi, DNA, viral nucleic acids, and / or exosomes.

10. The composition according to any one of claims 1 to 9, wherein the administration is performed within the wound, adjacent to the wound, and / or on the surface of the wound.

11. The composition according to any one of claims 1 to 9, wherein the administration to the individual is systemic administration.

12. The composition according to any one of claims 1 to 11, further comprising administering one or more adjuvants to an individual in the method described above.

13. The composition according to claim 12, wherein the adjuvant is based on a chemical, mechanical, viral, genetically modified component, or bacterial product.

14. The composition according to any one of claims 1 to 13, wherein the individual has diabetes, cancer, or is immunologically deficient.

15. The composition according to any one of claims 1 to 14, wherein the wound is chronic.

16. The composition according to any one of claims 1 to 15, wherein the wound is a cut, abrasion, bedsore, burn, or the individual has one or more of these.

17. The composition according to any one of claims 1 to 16, wherein the administration is performed once.

18. The composition according to any one of claims 1 to 16, wherein the administration is performed more than once.

19. The composition according to any one of claims 1 to 16, wherein the administration is performed daily, twice a day, three times a day, or four times a day.

20. The composition according to any one of claims 1 to 19, wherein one or more antibiotics are administered to the individual.

21. The composition according to claim 20, wherein one or more antibiotics are administered systemically to the individual.

22. The composition according to claim 20, wherein one or more antibiotics are administered locally to the individual.

23. The composition according to any one of claims 1 to 22, wherein a composition comprising fibroblasts and / or fibroblast-derived products is administered to or at a wound, and a cover is then placed over at least a portion of the wound.

24. The composition according to claim 23, wherein the cover functions as a barrier covering at least a portion of the wound to prevent fibroblasts and / or fibroblast-derived products from leaving the wound.

25. The composition according to claim 23 or 24, wherein the cover includes a protective piece of material.

26. The composition according to any one of claims 23 to 25, wherein the cover is a bandage.

27. The composition according to claim 26, wherein the bandage is a liquid bandage or a solid bandage.

28. A composition for use in a method for promoting the healing of one or more wounds in an individual, the composition comprising fibroblasts and / or fibroblast-derived products, The method comprises the step of administering a therapeutically effective amount of the composition to an individual.

29. The composition according to claim 28, wherein the fibroblast-derived product comprises extracellular vesicles, exosomes, microvesicles, apoptotic bodies, and / or fragments or biological components of fibroblasts.

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