Fibroblast compositions for treating aphthous ulcers

Abstract

Disclosed herein, in some aspects, are methods and compositions for treatment of aphthous ulcers, such as during cancer treatment. In some embodiments, the methods encompass fibroblasts, modified fibroblasts, and / or fibroblast derivatives, including in particular forms, for the treatment of aphthous ulcers, such as during cancer treatment. In some embodiments, fibroblast exosomes, lysate and / or conditioned media are provided in the form of lozenges to an individual undergoing cancer treatment.

Description

FIBROBLAST COMPOSITIONS FOR TREATING APHTHOUS ULCERSTECHNICAL FIELD AND BACKGROUNDI. Claim of Priority

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 666435 filed on July 1, 2024, incorporated by reference herein.II. Technical Field

[0002] Aspects of this disclosure relate to at least the fields of cell biology, molecular biology, immunology, and medicine.III. Background

[0003] Difficult-to-heal wounds, such as chronic wounds, are persistent, time-consuming, and costly to treat. In addition, injury to the skin due to diseases such as diabetes or cancer, cuts, abrasions, bedsores, or burns can have a lasting impact on a patient's physical, emotional, and psychological well-being. While some injuries caused to the skin can heal normally and quickly, certain underlying health aspects, such as age, health status, and certain diseases, can adversely impact the intricately orchestrated wound healing process, thereby requiring external intervention to heal properly.

[0004] Aphthous ulcers, also known as canker sores, represent a common and painful sore, and can be a complication experienced by individuals with healing complications, such as patients undergoing cancer treatment, particularly chemotherapy and radiation therapy. These ulcers can impair a patient's quality of life and may lead to treatment interruptions or modifications, thereby impacting overall treatment outcomes. As a consequence, aphthous ulcer complications represent a significant burden on the healthcare system that impacts the quality of life of patients, including those with non-healing wounds.

[0005] Aphthous ulcer healing follows an intricately orchestrated process of hemostasis, inflammation, proliferation, epithelialization, and remodeling confined to the injury location (Bainbridge, P., J Wound Care, 2013. 22(8): p. 407-8, 410-12). Conventional treatments for aphthous ulcers often provide limited relief and are not specifically tailored to address the underlying biological mechanisms contributing to ulcer formation and persistence (Sroussi, H.Y., et al., Cancer Med, 2017. 6(12): p. 2918-2931). Patients undergoing cancer therapy have reduced healing capabilities and compromised immune systems amplifying the complications of aphthous ulcers. Therefore, a new strategy to treat aphthous ulcers in patients undergoing cancer therapy is urgently needed.

[0006] The present disclosure satisfies a long felt need in the art for methods for treatment of aphthous ulcers in individuals, including those undergoing cancer therapy.SUMMARY

[0007] The methods described herein provide a novel and promising approach to treating aphthous ulcers thereby addressing a significant unmet clinical need, by utilizing fibroblast and / or fibroblast-derived factors for the ulcers. In some aspects, the fibroblast and / or fibroblast-derived factors produce one or more therapeutic factors and in some cases allow for delivering the therapeutic factor(s) in a localized manner. In some aspects, an individual with aphthous ulcers, or an individual susceptible thereto, include individuals that have cancer or are being treated for cancer. The disclosure provides for improving the management of aphthous ulcers and enhancing patient outcomes, such as in the oncology setting.

[0008] The present disclosure describes methods using fibroblasts and / or fibroblast- derived factors for treating any type of aphthous ulcers, including those occurring during cancer treatment. In some embodiments, the method involves culturing fibroblasts to allow for the production of fibroblast-derived factors, extracting and / or concentrating the factors, and delivering them in a means capable of inducing benefit to the individual. In some embodiments, the fibroblasts are derived from a pluripotent cell, e.g., an induced pluripotent stem cell, a pluripotent stem cell, an embryonic stem cell, and / or a non-embryonic stem cell. In some embodiments, the fibroblast is a trans-differentiated fibroblasts. In some embodiments, the fibroblasts are cultured as organoids. Such benefits may include reducing pain of the ulcer(s), reducing size of the ulcer(s), reducing number of the ulcer(s), facilitating partial or complete healing of the ulcer(s), reducing the risk of having one or more occur, a combination thereof, and so forth. In specific embodiments, the fibroblasts and / or fibroblast-derived factors are provided to the individual, such as through the administration of lozenges, films, lollipop-like structure, chewing gum, dissolvable tablet, and / or mouthwash.

[0009] The methods disclosed herein offers several advantages over existing treatments for aphthous ulcers. In some embodiments, the fibroblasts and / or fibroblast-derived factors are filtered, enriched, lysed, extracted, and / or concentrated. In some embodiments, the fibroblasts and / or fibroblast-derived factors (regardless of whether or not they have been filtered, enriched, lysed, extracted, and / or concentrated) are formulated into a lozenge, film, mouthwash, lollipoplike structure, chewing gum, or dissolvable tablet that improves delivery of factors to the ulcer or the site of ulcer formation. The localized delivery of the formulation via lozenges or other oral delivery carriers ensures direct contact with the ulcerated tissue, maximizing therapeuticefficacy while minimizing systemic side effects. Efficient delivery of the factors derived from fibroblasts enhances wound healing and ameliorates recurrence of aphthous ulcers.

[0010] In some embodiments, methods comprise activated or inactivated fibroblasts and / or derived factors thereof, e.g., exosomes, lysates, and / or apoptotic bodies. In some embodiments, the factors are delivered directly in, on, or around an ulcerous aphthous wound. In some embodiments, the derived factors comprise at least (a) extracellular matrix proteins, such as laminins, fibronectins, collagens, and elastic fibers; (b) growth factors and cytokines for wound healing; and / or (c) inhibitors of metalloproteinases. In specific embodiments, the cytokine is selected from the group consisting of bFGF, VEGF, HGF, PDGF, TGF-pi, KGF, IFN-gamma, TNF-alpha, interleukin(IL)-l, IL-6, IL-7, IL-8, IL-12, IL-15, IL-17, IL-33, and a combination thereof. In some embodiments, the therapeutic factors comprise factors derived from fibroblasts, fibroblast organoids, from culture of fibroblasts with other cells, or from culture of fibroblast organoids with other cells. In some embodiments, therapeutic factors comprise extracellular matrix factors, vasoconstriction regulating factors, antimicrobial factors, extracellular remodeling factors, growth factors, cytokines, inflammation modulating factors, chemokines, and / or clotting factors, for example, but not limited to collagens, glycosaminoglycans, hyaluronic acid, elastin, laminins, tenascins, fibronectin, endothelin, catecholamines, epinephrine, norepinephrine, prostaglandins, bradykinin, fibrinopeptide, serotonin, histamine, thromboxane A2, myeloperoxidase, azurocidin, lysozyme, bacterial permeability increasing protein, cathepsin G, elastase, protease 3, human cationic antimicrobial protein 18, lactoferrin, matrix metalloprotease 8, collagenase 2, chymase, tryptase, matrix metalloproteases, tissue inhibitor of metalloproteinases- 1, -2, -3, angiopoietins, FGF, KGF, VEGF, PDGF, TGF, EGF, IGF, TNF-alpha, IL-6, IL 1 -beta, INF-alpha, INF-beta, IL-2, IL-4, IL-8, IL- 10, IL-7, hyaluronan, osteopontin, periostin, vitronectin, angiotensin, CCN2, Cx43, CXCL8, CXCL1, CXCL12, CXCL2, RANTES, MCP, MIPs, lymphotactin, CCL2, CXCL10, fibrinogen, von Willebrand factor, collagen, fibrin, biglycan, decorin, versican, heparan sulfate, tenascin C, uric acid, S100 protein, ATP, F-actin, cyclophilin A, histones, HMGB1, IL-1 alpha, IL-33, SAP130, DNA, RNA, mtDNA, formyl peptide, mROS, calreticulin, defensins, cathelicidin, eosinophil-derived neurotoxin, granulysin, syndecans, glypicans, mast cell protease-4 or -5, CCN1, stromal derived factor-1 (SDF-1 / CXCL12), monocyte chemoattractant protein- 1 (MCP-1), Tie-2, SKINTs, CD 100, ICAM-1, VCAM-1, P-selectin, E-selectin. In some embodiments, the derived factors may recruit cells needed for the healing process while protecting the wound site and preventing infection. In some embodiments, the derived factors may promote proliferation and differentiation of epithelial cells in the mouth.

[0011] In some embodiments, fibroblasts for use in any methods of the disclosure may be exposed to one or more compositions and / or conditions to enhance their use in wound healing (“activated fibroblasts”). In some embodiments, the fibroblasts are activated prior to, or during, use for methods of the disclosure. In specific embodiments, fibroblasts are activated in vitro to produce activated fibroblast-derived factors. In some embodiments, fibroblasts are activated by exposure to platelets, neutrophils, monocytes, macrophages (e.g., Ml, M2, M2a), dendritic cells (e.g., Langerhans cells), T cells (e.g., aP T cells, y5 T cells, T helper cells, regulatory T cells, killer T cells), endothelial cells, pericytes, hematopoietic progenitor cells, epidermal cells, epidermal stem cells, smooth muscle cells, lymphocytes, mast cells, NK cells, adipose stromal cells, immune cells, keratinocytes, basal epithelial cells, myofibroblasts or derivatives therefrom. In some embodiments, activated fibroblasts have modified nucleic acids, cytokines, chemokines, growth factors, and / or exosomes and / or production thereof compared to nonactivated fibroblasts. In some embodiments, the fibroblast cells may be activated, such as having activated or engineered surface markers, nucleic acid modification, and / or expression or excretion of one or more chemokines, one or more cytokines, exosomes, and / or one or more growth factors. In some embodiments, the fibroblast may be activated by one or more chemical agents, RNA, micro RNA, RNAi, DNA, viral nucleic acid, and / or exosomes. In specific embodiments, the cytokine is selected from the group consisting of IFN-gamma, TNF-alpha, interleukin (IL)-l, IL-6, IL-7, IL-8, IL-12, IL-15, IL-17, IL-33, and a combination thereof. In specific embodiments, the growth factor is selected from the group consisting of FGF-1, VEGF, HGF, KGF, EPG and a combination thereof. In specific embodiments, the cells are activated following exposure to human platelet-rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, human serum, serum replacement, or a combination thereof. In specific embodiments, the fibroblasts are activated following exposure to hypoxia. The hypoxia may be 0.1%- 10%, 0.1%-5%, 0.1%-2.5%, or 0.1%-l% oxygen, for example. In specific embodiments, the hypoxia occurs for a period of time that is at least or no more than between about 30 minutes to about 3 days, although in some cases, it may be less than 30 minutes or greater than 3 days. The fibroblasts may be exposed to one or more agents prior to and / or during exposure to hypoxia, carbon monoxide, or a combination thereof. In some embodiments, the activated fibroblasts are used to generate activated fibroblast-derived factors. In some embodiments, cells that are used in the methods disclosed herein have not been activated.

[0012] In some embodiments, the introduction of fibroblasts, activated fibroblasts, fibroblast-derived factors, and / or activated fibroblast-derived factors into, on top of, and / or adjacent to the wound site results in (1) tissue differentiation; (2) cell recruitment anddifferentiation of stems cells at the wound site; (3) expansion of already locally present keratocytes, basal epithelial cells, myofibroblasts, endothelial cells, adipose cells, and / or dermal fibroblasts; and / or (4) attraction of other cells or biologic processes to aid in the healing, all for the purpose of initiating or maintaining wound healing. In some embodiments, the use of fibroblasts or fibroblast-derived factors promotes angiogenesis, repair, and regeneration in a wound site and surrounding tissue.

[0013] In some embodiments, fibroblasts, fibroblast-derived factors, or activated forms thereof are used with one or more adjuvants to activate the immune system and initiate and or maintain the wound-healing process. In some embodiments, adjuvants may be chemical, viral, bacterial, and / or genetically modified.

[0014] In specific embodiments, encapsulated RNA, microRNA, or RNAi, and / or fibroblast-derived exosomes or other microvesicles are utilized for any of the methods described herein.

[0015] In some embodiments, the individual is provided an effective amount of fibroblasts, fibroblast-derived factors, or activated forms thereof as a preventative of the ulcers. In such cases the individual may have a history of recurring ulcers, although in other cases the individual does not. In specific embodiments, the individual has a cut or injury inside their mouth and is provided an effective amount of fibroblasts, fibroblast-derived factors, or activated forms thereof for preventing development of an ulcer at and / or near the cut site or injury. In some embodiments, the individual has one or more apparatuses in the oral cavity, such as orthodontic apparatuses including braces or retainers (removable or permanent), or the individual may have dentures or experienced overzealous brushing.

[0016] Aspects of the present disclosure address certain needs by providing at least methods useful in cell biology and regenerative biology.

[0017] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodimentsdiscussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0018] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0019] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DETAILED DESCRIPTIONI. Definitions

[0020] It is to be understood that the present disclosure is not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0022] The practice of the present disclosure may employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds.(1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition, all of which are incorporated herein by reference in their entirety.

[0023] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. It is to be inferred without explicit recitation and unless otherwise intended, that when the present technology relates to a polypeptide, protein, polynucleotide, cell, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.

[0024] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0025] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0026] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0027] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form ofcontaining, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0029] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0030] The term "individual", as used herein, refers to a human or animal that may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions. An individual may comprise any age of a human or non-human animal and therefore includes both adults and juveniles (i.e., children) and infants. It is not intended that the term "individual" connotes a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. The term “subject” or “individual” may be used interchangeably and refers to any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0031] The term "administered" or "administering", as used herein, refers to any method of providing a composition to an individual such that the composition has its intended effect on the patient. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, applicator gun, syringe, etc. A second exemplary method of administering is by a direct mechanism such as, local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository, etc.

[0032] As used herein, “agent” refers to nucleic acids, cytokines, chemokines, transcription factors, epigenetics factors, growth factors, or hormones.

[0033] As used herein, “allogeneic” refers to tissues or cells from another body that in a natural setting are immunologically incompatible or capable of being immunologically incompatible, although from one or more individuals of the same species.

[0034] As used herein, “autologous” refers to tissues or cells that are derived or transferred from the same individual's body (e.g., autologous blood donation; an autologous bone marrow transplant).

[0035] As used herein, “xenogeneic” refers to tissues or cells from a species different from the individual.

[0036] Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pre-treatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, “treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the incidence, recurrence, persistence, and / or symptoms of aphthous ulcers is considered to be a treatment if there is a detectable reduction in the incidence, recurrence, persistence, and / or symptoms of aphthous ulcers when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition. In specific embodiments, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.

[0037] Cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent, or actively dividing. In a cell culture, cells are grown and maintained at an appropriate temperature, typically a temperature of about 37 °C and under an atmosphere typically containing oxygen and CO2. Culture conditions may vary widely for each cell type though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly varied factor in culture systems is the growth medium. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0038] The term “organoid” as used herein refers to self-organized, three-dimensional tissue cultures that are derived from cells that have self-renewal and differentiation capacities. In specific cases, the term applies to organoids having specific shapes or substantially similar to specific shapes, such as spheroids. In specific cases, the cells that have self-renewal and differentiation capacities are fibroblasts.

[0039] As used herein, the term “activated fibroblasts” refers to any kind of fibroblasts treated with one or more stimuli or agent(s) capable of inducing one or more alterations in the fibroblast: metabolic, immunological, epigenetic, growth factor-secreting, surface marker expression, and production and excretion of microvesicles. Examples of agents include at least ADP, CXCL4, PDGF, TGF-P, IL-1, TNF-a, ROS, NO, AMP, IL-17, IL22, dsRNA 1.25D3, IL-8, CXCL1-8, Activin A, P -defensins, cathelici din, E-selectin, P-selectin, KGF, VEGF, IGF, IL- 10, IL-4, and IL-13.

[0040] The term “fibroblast-derived factors” or “fibroblast-derived materials” as used herein refer to fibroblast cell fragments, vesicles or exosomes secreted from fibroblasts, fibroblast conditioned media, fibroblast lysate, a combination thereof and / or a purified, filtered, enriched, and / or concentrated fraction thereof.

[0041] The term “apoptotic body” or “apoptotic bodies” as used herein refers to cell fragments produced by blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and / or chromosomal DNA fragmentation as a result of cell apoptosis.

[0042] The term “conditioned media” or “supernatant” as used herein is refers to the media harvested from cultured cells.

[0043] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0044] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.II. Aphthous Stomatitis

[0045] Aphthous stomatitis is a condition that causes ulcers inside the mouth, typically on the buccal mucosa, lateral tongue, soft palate, and the floor of the mouth. Aphthous ulcers are characterized by an erythematous halo and a white pseudomembrane. There are three types of aphthous ulcers conditions classified by their size and severity. Minor aphthous ulcers are less than 1 cm in diameter, appear as a small cluster of 1-6 ulcers, and usually heal within 2 weeks without scarring. Major aphthous ulcers are more than 1 cm in diameter, may take up to 10 weeks to heal, and often heal with scarring. Herpetiform ulcers are characterized by clusters of numerous ulcers, e.g., up to about 100 ulcers, of small size, i.e., about 2-3 mm. Herpetiform ulcers may coalesce to form larger ulcers and typically heal within 1-2 weeks. Recurrent aphthous ulcers may last days to months. Depending on the degree of severity and recurrence, aphthous ulcers can affect oral functions (e.g., nutrition) and quality of life (e.g., pain, discomfort, bleeding, infection, or weight loss). In some instances, complications from aphthous ulcers include disruption of ongoing medical treatment and / or increased risk of infections and septicemia.

[0046] The etiology of aphthous ulcers can be multifactorial and may be specific to the individual. Aphthous ulcers can be caused by viral (e.g., herpes simplex virus, coxsackie virus, Epstein-Barr virus, or human immunodeficiency virus), bacterial (e.g., streptococcus, actinomyces, syphilis, tuberculosis), or fungal infections (e.g., Candida). In many cases, aphthous ulcers are caused by autoimmune disorders (e.g., oral lichen planus, systemic lupus erythematosus, vesiculobullous disease, erythema multiform, or benign migratory glossitis). In some cases, aphthous ulcers may be caused by gastrointestinal disorders (e.g., Chron’s disease, ulcerative colitis, celiac disease, inflammatory bowel disease, or irritable bowel syndrome). In some cases, aphthous ulcers are caused by mechanical injury (e.g., cuts, abrasions, bites, etc.), hormone imbalances (e.g., stress, menstruation, pregnancy, menopause, or hormone therapy), or sensitivity to topical agents, foods, alcohol, tobacco, drugs, and / or medicaments (e.g., anticancer medicaments).

[0047] It is estimated that up to about 90% of individuals undergoing cancer therapy develop aphthous ulcers (Brown and Gupta, JCO Oncol Pract. 2020 Mar; 16(3): 103-109.). Several types of cancer therapy, e.g., radiotherapy, chemotherapy, or immunotherapy, cause aphthous ulcers by inducing epithelial cell damage and / or impairing immune system function. Recurrence and severity of cancer therapy-associated aphthous ulcers have been shown to increase with dosage or duration of the cancer therapy (Sonis et al., Cancer. 2010 Jan 1; 116(l):210-5; Sroussi et al., Cancer Med, 2017. 6(12): p. 2918-2931; Lalla et al., Cancer. 2014 May 15; 120(10): 1453-61). Aphthous ulcers in cancer patients can lead to de-escalation of cancer therapy, superinfections, and septicemia.

[0048] Current protocols for the treatment of aphthous ulcers are limited, primarily due to the location of the ulcers. Typical ointments or products to treat ulcers in other tissues, e.g., skin, cannot always be applied inside the mouth and / or are ineffective due to the presence of saliva, enzymes, and / or cells. Currently, treatment for aphthous ulcers is preventative or palliative, such as by increased attention to oral care (e.g., regular use of mouthwash), pain management (e.g., application of lidocaine or morphine), and administration of antiinflammatory drugs until the tissue regenerates naturally. In some instances, corticosteroid and immunosuppressants may be used. However, for patients undergoing cancer therapy, who may have impaired tissue regeneration and / or a compromised immune system, the current approaches to manage aphthous ulcers are insufficient.

[0049] In some embodiments, there is a novel method to treat aphthous ulcers in an individual, and the individual may or may not be undergoing cancer therapy, the method comprising providing fibroblasts and / or fibroblast-derived factors to the affected buccal area. They may or may not be provided in the form of lozenges, films, lollipop-like structure, chewing gum, dissolvable tablet, and / or mouthwash. In some embodiments, the individual is undergoing chemotherapy, radiotherapy, immunotherapy, oncolytic virus therapy, hormone therapy, surgery, and / or a combination thereof. In some embodiments, the individual has minor, major, and / or recurrent aphthous ulcers. In some embodiments, the aphthous ulcers have persisted for more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more weeks before treatment with a composition described herein. In some embodiments, the fibroblasts and / or fibroblast-derived factors are administered before, during, or after a cancer therapy. In some embodiments, the fibroblasts and / or fibroblast-derived factors are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a day. In some embodiments, the fibroblasts and / or fibroblast-derived factors are administered throughout the entire duration of the cancer therapy. In some embodiments, the fibroblasts and / or fibroblast-derived factorsare administered ad libitum. In some embodiments, the individual is provided an effective amount of fibroblasts and / or fibroblast-derived factors at the onset of therapy for cancer, regardless of whether or not they presently have an ulcer. Such therapy may or may not be provided for the duration of the therapy.

[0050] In certain aspects, an individual is treated or prevented from having ulcers, such as from a minor injury to the mouth from dental work, overzealous brushing, sports mishaps, or an accidental cheek bite. In specific aspects, the individual is a user of toothpastes and / or mouth rinses that comprise sodium lauryl sulfate. In some aspects, the individual has one or more types of food sensitivities, such as to chocolate, coffee, strawberries, eggs, nuts, cheese, or spicy or acidic foods. In some aspects, the individual is a consumer of tobacco. In some aspects, the individual as a consumer of alcohol. In some aspects, the individual has a diet lacking in vitamin B-12, zinc, folate (folic acid) and / or iron. In certain aspects, the individual has an allergic response to certain bacteria in the mouth. In some aspects, the individual is infected with Helicobacter pylori. In some aspects, the individual has a fungal infection, e.g., Candida infection. In some embodiments, the individual is menstruating or about to menstruate. The individual may be experiencing emotional stress.III. Fibroblasts

[0051] Fibroblasts, modified fibroblasts, activated fibroblasts, fibroblast organoids, and / or products derived therefrom are utilized in methods disclosed herein for wound healing, including to initiate and / or maintain wound healing of aphthous ulcers.

[0052] In some embodiments, fibroblasts and / or fibroblast derived products are used as biologic components for wound healing, including to initiate and / or maintain wound healing. In some embodiments, the fibroblasts and / or fibroblast derived products may be activated prior to, or during, use for methods disclosed herein.

[0053] In specific embodiments, one or more compositions of the disclosure include one or more of fibroblasts, exosomes from fibroblasts, lysate from fibroblasts, apoptotic bodies from fibroblasts, conditioned media from fibroblasts, fibroblast-derived products, or any combination thereof. In some embodiments, one or more distinct compositions may be utilized on the same wound, e.g., aphthous ulcer. In specific embodiments, encapsulated RNA, microRNA, or RNAi, fibroblast-derived exosomes and / or other fibroblast-derived microvesicles may be utilized for any of the methods described herein.

[0054] In various embodiments, the introduction of a composition described herein into, on top of, and / or adjacent to the wound, e.g., an aphthous ulcer, results in (1) tissue differentiation; (2) cell recruitment and differentiation of stems cells at the wound site; (3) expansion of already locally present keratocytes, basal epithelial cells, endothelial cells, myofibroblasts, and / or dermal fibroblasts; and / or (4) attraction of other cells or biologic processes to aid in the healing, all for the purpose of initiating or maintaining wound healing.

[0055] In some embodiments, a biological component is used with one or more adjuvants to activate the immune system and initiate and / or maintain the wound-healing process. These adjuvants could be chemical, viral, bacterial, and / or genetically modified components.

[0056] Embodiments of the disclosure include in vitro methods of producing activated or non-activated fibroblasts, exosomes from fibroblasts, lysate from fibroblasts, apoptotic bodies from fibroblasts, and / or fibroblast-derived factors for use in any of the methods described herein. The fibroblasts, exosomes from fibroblasts, lysate from fibroblasts, apoptotic bodies from fibroblasts, and / or fibroblast-derived factors may be autologous, allogeneic, syngeneic, or xenogeneic with respect to the individual.

[0057] In some embodiments, the fibroblasts may be derived from another cell type, for example, through differentiation or trans-differentiation. In some embodiments, fibroblasts are generated in vitro from a pluripotent cell, e.g., induced pluripotent stem cells, embryonic stem cells, non-embryonic stem cells, etc. In some embodiments, a pluripotent cell is differentiated into a fibroblasts for use in methods of the disclosure. In some embodiments, pluripotent cells are grown in irradiated feeder layers of mouse embryonic fibroblasts, with normal human keratinocyte medium (3: 1 Dulbecco's modified Eagle's medium (DMEM):F12 (Invitrogen, Carlsbad, CA), 5% FCII (Hyclone, Logan, UT), 0.18 mM adenine, 8 mM HEPES, 0.5 pg / mL hydrocortisone, 1010M cholera toxin, lO ng / mL epidermal growth factor, and 5 pg / mL insulin), with bone morphogenetic protein 4 (MBP-4), on collagen-coated plates, and / or any other condition that induces differentiation of a pluripotent cell into fibroblasts (see e.g., Hewitt KJ, Shamis Y, Carlson MW, Aberdam E, Aberdam D, Garlick JA. Three-dimensional epithelial tissues generated from human embryonic stem cells. Tissue Eng Part A. 2009 Nov;15(l 1):3417-26, which is incorporated in its entirety herein by reference).A) Fibroblast Isolation

[0058] The fibroblasts and / or fibroblast derivatives considered in this disclosure may be generated, in one or more embodiments, by outgrowth from a biopsy of the recipient's ownskin (in the case of autologous preparations), or skin of healthy donors (for allogeneic preparations). In some embodiments, the fibroblasts of the present disclosure are from a xenogeneic source with respect to an individual. In some embodiments, fibroblasts may come from infants, children, adolescents, and / or adults. In some embodiment, fibroblasts are transfected and / or transduced with heterologous genes for enhanced growth and overcoming of the Hayflick limit. This may or may not occur subsequent to derivation of cells and expansion in culture using standard cell culture techniques.

[0059] The fibroblasts of the present disclosure may be from any source or tissue including fibroblasts derived from skin, adipose tissue, bone marrow, umbilical cord, Wharton’s jelly, omentum, peripheral blood, perivascular tissue, adipose tissue, placental tissue, amniotic membrane, omentum, dental pulp, fallopian tube tissue, hepatic tissue, renal tissue, cardiac tissue, tonsillar tissue, testicular tissue, ovarian tissue, neuronal tissue, auricular tissue, colonic tissue, submucosal tissue, hair follicle tissue, pancreatic tissue, skeletal muscle tissue, subepithelial umbilical cord tissue, foreskin tissue, mobilized peripheral blood, or a combination thereof. Mobilized peripheral blood may be obtained by administering to an individual a composition that generates mobilized peripheral blood, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM- CSF), FMS-like tyrosine kinase 3 (Flt-3) ligand, plerixafor (including Mozobil™), hyperbaric oxygen, ozone therapy, or a combination thereof.

[0060] The fibroblasts and / or fibroblast derivatives may be obtained by any suitable manner. As one example, skin tissue (dermis and epidermis layers) may be biopsied from an individual’s post-auricular area. In some embodiments, the starting material is comprised of three 3 -mm punch skin biopsies collected using standard aseptic practices. The biopsies may be placed into a vial containing sterile phosphate buffered saline (PBS) and shipped in a 2-8 °C. refrigerated shipper back to a manufacturing facility. In some embodiments, after arrival at the manufacturing facility, the biopsy is inspected and, upon acceptance, transferred directly to the manufacturing area. Upon initiation of the process, the biopsy tissue is then washed prior to fibroblast isolation from the tissue sample.

[0061] In some embodiments, fibroblast cells are isolated from a sample or biopsy of bodily tissue by enzymatic digestion, mechanical separation, filtration, centrifugation, or combinations thereof. The number and quality of the isolated fibroblast cells can vary depending, e.g., on the quality of the tissue used, the compositions of perfusion buffer solutions, and the type and concentration of enzyme. Frequently used enzymes include, but are not limited to, collagenase, pronase, trypsin, dispase, hyaluronidase, thermolysin and pancreatin, andcombinations thereof. Collagenase is most commonly used, often prepared from bacteria e.g., from Clostridium histolyticum), and may often consist of a poorly purified blend of enzymes, which may have inconsistent enzymatic action. Some of the enzymes exhibit protease activity, which may cause unwanted reactions affecting the quality and quantity of viable / healthy fibroblast cells. It is understood by those of skill in the art to use enzymes of sufficient purity and quality to obtain viable fibroblast cell populations.

[0062] In some embodiments, a collagenase enzyme solution (e.g., Liberase™) is added to the tissue sample without mincing and incubated at 37.0±2 °C. for about one hour. Time of biopsy tissue digestion is a critical process parameter that can affect the viability and growth rate of cells in culture. Liberase is a collagenase / neutral protease enzyme cocktail obtained formulated from Lonza Walkersville, Inc. (Walkersville, Md.) and unformulated from Roche Diagnostics Corp. (Indianapolis, Ind.). Alternatively, other commercially available collagenases may be used, such as Serva Collagenase NB6 (Helidelburg, Germany). After digestion, Initiation Growth Media (IMDM, GA, 10% Fetal Bovine Serum (FBS)) is added to neutralize the enzyme, cells are pelleted by centrifugation and re-suspended in 5.0 mL Initiation Growth Media. Alternatively, centrifugation is not performed, with full inactivation of the enzyme occurring by the addition of Initiation Growth Media only. Initiation Growth Media is added prior to seeding of the cell suspension into a T-175 cell culture flask for initiation of cell growth and expansion. A T-75, T-150, T-185 or T-225 flask can be used in place of the T-175 flask. Cells are incubated at 37±2.0 °C. with 5.0±1.0% CO2 and fed with fresh Complete Growth Media every three to five days. All feeds in the process are performed by removing half of the Complete Growth Media and replacing the same volume with fresh media. Alternatively, full feeds can be performed. Cells should not remain in the T-175 flask greater than 30 days prior to passaging. Confluence is monitored throughout the process to ensure adequate seeding densities during culture splitting. When cell confluence is greater than or equal to 40% in the T-175 flask, they are passaged by removing the spent media, washing the cells, and treating with Trypsin-EDTA to release adherent cells in the flask into the solution. Cells are then trypsinized and seeded into a T-500 flask for continued cell expansion. Alternately, one or two T-300 flasks, One Layer Cell Stack (1 CS), One Layer Cell Factory (1 CF) or a Two Layer Cell Stack (2 CS) can be used in place of the T-500 Flask. Cells may be incubated at 37±2.0 °C. with 5.0±1.0% CO2 and passaged every three to five days in the T-500 flask and every five to seven days in the ten layer cell stack (10 CS). Cells should not remain in the T-500 flask for more than 10 days prior to passaging. Quality Control (QC) release testing for safety includes sterility and endotoxin testing. When cell confluence in the T-500flask is about 95%, cells are passaged to a 10 CS culture vessel. Alternately, two Five Layer Cell Stacks (5 CS) or a 10 Layer Cell Factory (10 CF) can be used in place of the 10 CS. Passage to the 10 CS is performed by removing the spent media, washing the cells, and treating with Trypsin-EDTA to release adherent cells in the flask into the solution. Cells are then transferred to new 10 CS. Additional Complete Growth Media is added to neutralize the trypsin and the cells from the T-500 flask are pipetted into a 2 L bottle containing fresh Complete Growth Media. The contents of the 2 L bottle are transferred into the 10 CS and seeded across all layers. Cells are then incubated at 37±2.0 °C. with 5.0±1.0% CO2 and fed with fresh Complete Growth Media every five to seven days. Cells should not remain in the 10 CS for more than 20 days prior to passaging. In one embodiment, the passaged fibroblasts are rendered substantially free of immunogenic proteins present in the culture medium by incubating the expanded fibroblasts for a period of time in protein free medium. When cell confluence in the 10 CS is about 95% or more, cells may be harvested for use in any of the methods described herein. Harvesting is performed by removing the spent media, washing the cells, treating with Trypsin-EDTA to release adherent cells into the solution, and adding additional Complete Growth Media to neutralize the trypsin. Cells are collected by centrifugation, resuspended, and in-process QC testing performed to determine total viable cell count and cell viability.

[0063] Morphology is evaluated at each passage and prior to harvest to monitor the culture purity throughout the process. Morphology is evaluated by comparing the observed sample with visual standards for morphology examination of cell cultures. The cells display typical fibroblast morphologies when growing in cultured monolayers. Cells may display either an elongated, fusiform or spindle appearance with slender extensions, or appear as larger, flattened stellate cells which may have cytoplasmic leading edges. A mixture of these morphologies may also be observed. Fibroblasts in less confluent areas can be similarly shaped, but randomly oriented.

[0064] The presence of keratinocytes in cell cultures is also evaluated. Keratinocytes appear round and irregularly shaped and, at higher confluence, they appear organized in a cobblestone formation. At lower confluence, keratinocytes are observable in small colonies.B) Fibroblast Markers

[0065] In some embodiments, fibroblast cells of the present disclosure are identified and characterized by their expression of specific marker genes, such as cell-surface markers. Detection and isolation of these cells can be achieved, e.g., through flow cytometry, FACS,immunohistochemistry, immunofluorescence, imaging, ELISA, and / or magnetic bead separation. Reverse-transcription polymerase chain reaction (RT-PCR) can also be used to monitor changes in gene expression. In certain embodiments, the markers used to identify and characterize the fibroblasts are selected from the group consisting of c-Kit (also known as CD117), has related family bHLH transcription factor with YRPW motif 1 (Heyl), a-smooth muscle actin (SMA), Vimentin (including intracellular or extracellular vimentin), Cyclin D2, Snail, E-cadherin, NK2 homeobox 5 (Nkx2.5), GATA binding protein 4 (GATA4), cluster of differentiation (CD) 105, CD271, CD90, CD29, CD73, CD44, CD10, CD13, CD44, Wilms’ tumor 1 (Wtl), CXC motif chemokine receptor 4 (CXCR-4), fibroblast growth factor 1 (FGF- 1) receptor, stage specific embryonic antigen 3 (SSEA-3), tumor necrosis factor alpha (TNF- a) receptor- 1, toll like receptor 4 (TLR4), receptor for acetylated end products (RAGE), hepatocyte growth factor (HGF), express octamer-binding transcription factor 4 (Oct-4), CD- 34, Kriippel-like factor 4 (KLF-4), Nanog, Sox-2, Rex-1 (also known as zinc finger protein 42), growth differentiation factor 3 (GDF-3), Stella (also known as developmental pluripotency-associated 3), or possess enhanced expression of GDF-11, and a combination thereof. In specific aspects, fibroblasts and mesenchymal stem cells (MSCs) are distinguishable from each other because MSCs do not express octamer-binding transcription factor 4 (Oct-4), CD-34, Kriippel-like factor 4 (KLF-4), Nanog, Sox-2, Rex-1 (also known as zinc finger protein 42), growth differentiation factor 3 (GDF-3), Stella (also known as developmental pluripotency-associated 3), or possess enhanced expression of GDF-11.

[0066] In specific cases the fibroblasts may possess the ability to inhibit TNF-alpha production from an activated macrophage. The fibroblasts may express a higher amount of T cell proliferation inhibitory activity as compared to bone marrow derived mesenchymal stem cells when cultured under similar or identical conditions. The fibroblasts may express, relative to bone marrow-derived mesenchymal stem cells, increased levels of M-CSF under identical culture conditions.

[0067] The fibroblasts may have the potential to differentiate into cells of a chondrogenic phenotype when cultured under chondrogenic inductive media. In specific cases, the fibroblasts are isolated from adipose tissue substantially free of blood that is capable of self-renewal and expansion in culture. The fibroblasts may have the potential to differentiate into cells of other phenotypes besides fibroblasts, e.g., osteoblasts, adipocytes, and / or chondrocytes.

[0068] The fibroblasts of the disclosure, in some embodiments, may be cultured in a liquid media, and they may possess some or all of the following characteristics: (i) expression or production of the one of more antigens comprising c-Kit (also known as CD 117), hes relatedfamily bHLH transcription factor with YRPW motif 1 (Heyl), a-smooth muscle actin (SMA), Vimentin (including intracellular or extracellular vimentin), Cyclin D2, Snail, E-cadherin, NK2 homeobox 5 (Nkx2.5), GATA binding protein 4 (GATA4), cluster of differentiation (CD) 105, CD271, CD90, CD29, CD73, CD44, CD10, CD13, CD44, Wilms’ tumor 1 (Wtl), CXC motif chemokine receptor 4 (CXCR-4), fibroblast growth factor 1 (FGF-1) receptor, stage specific embryonic antigen 3 (SSEA-3), tumor necrosis factor alpha (TNF-a) receptor-1, toll like receptor 4 (TLR4), receptor for acetylated end products (RAGE), hepatocyte growth factor (HGF), express octamer-binding transcription factor 4 (Oct-4), CD-34, Kriippel-like factor 4 (KLF-4), Nanog, Sox -2, Rex-1 (also known as zinc finger protein 42), growth differentiation factor 3 (GDF-3), Stella (also known as developmental pluripotency-associated 3), or possess enhanced expression of GDF-11; (ii) ability to differentiate into osteoblasts, adipocytes and / or chondrocytes; (iii) hypoimmunogenicity as observed by reduced ability to stimulate allogeneic T cell proliferation compared to epithelial cells; (iv) have active immune suppressive activity, such as determined by an ability to inhibit proliferation of an ongoing mixed lymphocyte reaction, or a combination thereof.

[0069] In specific embodiments, modified fibroblasts have one or more of the aforementioned characteristic(s), and / or products derived from fibroblasts come from fibroblasts that have one or more of the aforementioned characteristic(s).C) Fibroblast Culture

[0070] The methods of the disclosure comprise culturing fibroblast cells obtained from tissue samples, e.g., human samples. In particular embodiments, the populations of fibroblast cells are plated onto a substrate. In some embodiments, fibroblasts are plated onto a substrate which allows for adherence of cells thereto. This may be carried out, e.g., by plating the cells in a culture plate which displays one or more substrate surfaces compatible with cell adhesion. When the said one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, the term "plating onto a substrate which allows adherence of cells thereto" refers to introducing cells into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the substrate surface.

[0071] General principles of maintaining adherent cell cultures are well-known in the art. As appreciated by those skilled in the art, the fibroblast cells may be counted in order tofacilitate subsequent plating of the cells at a desired density. Where the cells after plating may primarily adhere to a substrate surface present in the culture system (e.g., in a culture vessel), the plating density may be expressed as number of cells plated per mm2or cm2of the said substrate surface. In practicing the disclosure, after plating of the fibroblasts, the cell suspension is left in contact with the adherent surface to allow for adherence of cells from the cell population to the substrate. In contacting fibroblasts to the adherent substrate, the cells may be advantageously suspended in an environment comprising at least a medium, in the methods of the disclosure typically a liquid medium, which supports the survival and / or growth of the cells. The medium may be added to the system before, together with or after the introduction of the cells thereto. The medium may be fresh, z.e., not previously used for culturing of cells, or may comprise at least a portion which has been conditioned by prior culturing of cells therein, e.g., culturing of the cells which are being plated or antecedents thereof, or culturing of cells more distantly related to or unrelated to the cells being plated.

[0072] The medium may be a suitable culture medium as described elsewhere in this specification. In some cases, the composition of the medium may have the same features, may be the same or substantially the same as the composition of medium used in the ensuing steps of culturing the attached cells. Otherwise, the medium may be different. In some embodiments, the cells from the fibroblast cell population or from tissue explants of the present disclosure, which have adhered to the substrate, preferably in the environment, are subsequently cultured for at least 7 days, for at least 8 days, or for at least 9 days, for at least 10 days, at least 11, or at least 12 days, at least 13 days or at least 14 days, for at least 15 days, for at least 16 days or for at least 17 days, or even for at least 18 days, for at least 19 days or at least 21 days or more. The term "culturing" is common in the art and broadly refers to maintenance and / or growth of cells and / or progeny thereof.

[0073] In some embodiments, the fibroblast cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some embodiments, the fibroblasts of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The tissue explants and fibroblasts may be cultured in the presence of a liquid culture medium. Typically, the medium will comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture fibroblasts herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove'sModified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the fibroblasts cultured. In some embodiments, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS, Lonza), 100 U / mL penicillin G, 100 mg / mL streptomycin, and 2 mmol / L L-glutamine (Sigma- Aldrich). Other embodiments may employ further basal media formulations, such as chosen from the ones above.

[0074] For use in the fibroblast culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., betamercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated (e.g., FBS).

[0075] The fibroblasts of the disclosure, when cultured in liquid media, may show upregulation of one or more genes involved in the cell cycle (e.g., cyclins and cyclin dependent kinases) and / or in DNA replication and / or purine metabolism when compared to standard or non-modified fibroblasts or mesenchymal stem cells. In particular embodiments, genes functionally active in cell adhesion / extracellular matrix (ECM)-receptor interaction, differentiation / development, TGF-P signaling, and TSP-l-induced apoptosis are downregulated in the fibroblasts of the disclosure, when compared to standard or non-modified fibroblasts or mesenchymal stem cells. Specific examples of TGF-beta associated signalinggenes, which are downregulated by the fibroblasts, include at least SMAD1, SMAD2, SMAD3, SMAD4, GITR (also known as TNF receptor superfamily member 18), programmed deathligand 1 (PD-L1), CD5, and / or CD31.

[0076] The fibroblasts of the disclosure may be cultured in media supplemented with platelet lysate (PL) and / or fetal calf serum (FCS), in some cases. In some cases, the fibroblasts are cultured in platelet lysate (PL) containing media. For example, 300 pL of fibroblasts may be cultured in 15 mL of PL supplemented medium in a T-75 flask or other adequate tissue culture vessels. After washing away the non-adherent cells, the adherent cells are also cultured in media that has been supplemented with platelet lysate. Thrombocytes are a well characterized human product that is widely used in clinics for individuals in need of blood supplement. Thrombocytes are known to produce a wide variety of factors, e.g. platelet derived growth factor subunit b (PDGF-BB), transforming growth factor beta (TGF-P), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF). In some embodiments, an optimized preparation of PL is used. This optimized preparation of PL is made up of pooled platelet rich plasmas (PRPs) from at least 10 donors (to equalize for differences in cytokine concentrations) with a minimal concentration of 3xl09thrombocytes / mL.

[0077] According to particular embodiments of the method of producing fibroblasts of the disclosure, fibroblasts are exposed to PL that was prepared either from pooled thrombocyte concentrates designed for human use (produced as TK5F from the blood bank at the University Clinic UKE Hamburg-Epp endorf, pooled from 5 donors) or from 7-13 pooled buffy coats after centrifugation with 200xg for 20 min. In particular cases, the PRP was aliquoted into small portions, frozen at -80 °C., and thawed immediately before use to produce PL. In particular embodiment, PL-containing medium is prepared freshly for each cell feeding. In a particular embodiment, medium is used that contains alpha-MEM as basic medium supplemented with 5 IU Heparin / mL medium and 5% of freshly thawed PL. The method of producing fibroblasts of the disclosure may use a method to prepare PL that differs from others according to the thrombocyte concentration and centrifugation forces, in some cases.

[0078] In some embodiment, cells are cultured in media at about 37 °C with approximately 5% CO2 under hypoxic conditions. In certain aspects, the hypoxic conditions are an atmosphere of 5% O2. In some situations, hypoxic culture conditions allow fibroblasts to grow more quickly. This allows for a reduction of days needed to grow the cells to 90-95% confluence. Generally, it reduces the growing time by about three days. In some embodiments, adherent cells are cultured in media at about 37 °C with approximately 5% CO2 under normoxicconditions, i.e. wherein the O2 concentration is the same as atmospheric O2, at approximately 20.9%. In some embodiments, the cells are grown to between 70 and 90% confluence. In some embodiments, once this level of confluence is reached, the cells may be enzymatically detached, for example using trypsin.

[0079] In certain embodiments, the population of cells that is isolated from the plate is between 85-95% fibroblasts. In other embodiments, the fibroblasts are greater than 95%, 96%, 97%, 98%, or 99% of the isolated cell population.

[0080] In some embodiments, a closed system may be used for generating and expanding the fibroblast of the disclosure from bone marrow of normal donors. This closed system may be a device to functionally expand cells ex vivo. In one specific embodiment, the closed system comprises: 1) a central expansion unit preferably constructed similarly to bioreactors with compressed (within a small unit), but extended growth surfaces; 2) media bags that can be sterilely connected to the expansion unit (e.g. by welding tubes between the unit and the bags) for cell feeding; and 3) electronic devices to operate automatically the medium exchange, gas supply and temperature. The advantages of the closed system in comparison to conventional flask tissue culture are the construction of a functionally closed system, i.e. the cell input and media bags are sterile welded to the system. This minimizes the risk of contamination with external pathogens and therefore may be highly suitable for clinical applications. Furthermore, this system can be constructed in a compressed form with consistently smaller cell culture volumes but preserved growth area. The smaller volumes allow the cells to interact more directly with each other which creates a culture environment that is more comparable to the in vivo situation of the bone marrow niche. Also, the closed system saves costs for the media and the whole expansion process.

[0081] The construction of the closed system may involve two sides: the cells are grown inside of multiple fibers with a small medium volume. In some embodiments, the culture media comprises growth factors for growth stimulation, and medium without expensive supplements is passed outside the fibers. The fibers are designed to comprise nanopores for a constant removal of potentially growth-inhibiting metabolites while important growth-promoting factors are retained in the growth compartment.

[0082] In certain embodiments, a closed system may be used in conjunction with a medium for expansion of fibroblast that does not contain any animal proteins, e.g., fetal calf serum (FCS). FCS has been connected with adverse effects after in vivo application of FCS-expanded cells, e.g., formation of anti-FCS antibodies, anaphylactic or arthus-like immune reactions or arrhythmias after cellular cardioplasty. FCS may introduce unwanted animal xenogeneicantigens, viral, prion and zoonose contaminations into cell preparations making new alternatives necessary.D) Fibroblast Organoids

[0083] Embodiments of the disclosure may utilize organoids. In specific embodiments, the organoids are produced such that they may (1) initiate or maintain extended time release of immune modulation, (2) initiate or maintain extended time release of activated or inactivated fibroblasts and / or fibroblast-derived material, and / or (3) initiate or maintain extended time release migration and proliferation to a targeted tissue or organ for any purpose, including for repair, regeneration and / or reversal of involution. The cell structure of the organoid, as opposed to a plurality of single cells, allows for enhanced cell contact inhibition, the fibroblast cells are naturally reduced in their proliferation rate and metabolism rate to minimize oxygen consumption and nutrient consumption. Methods of production or use of organoids are described in PCT / US2023 / 076365, which is incorporated herein by reference in its entirety.

[0084] As an initial step of the methods, or soon after an initial step of the methods, fibroblast cells are manipulated to form organoids (including spheroids) under appropriate suspension culture conditions. In some embodiments for fibroblast spheroid formation, a U / V bottom type ultra-low attachment culture plate or hanging drop method can be used, according to different conditions. For example, cell organoids of different sizes may be generated according to the density of fibroblasts in the medium. In some cases, surface tension on a substrate is manipulated to enhance formation of the organoids. In a specific case, a surface is treated with a hydrophobic material except for a hydrophilic area upon which individual cells or clumps of cells are placed to facilitate production of the organoid. In some cases, spheroids can be formed by forming a core-shell structure within a pipette tip (e.g., (1) making the core spheroid in a single pipette tip and culturing for 48 hours; (2) creating a second layer on the spheroids with cells in the same pipette tip and culturing for 48 hours, repeating as desired to add additional layers of cells and / or cell types to the spheroid; and (3) after the cells spheroids form in the pipette tip for 96 hours, collecting the cell spheroids from the pipette tip by ejecting the liquid containing the spheroid from inside the pipette tip, removing the liquid matrix using centrifugation or filtration, and collecting the cell spheroids). It is also possible to directly use ultra-low attachment culture plates or glass culture flasks to spread fibroblasts into vessels or culture dishes at high density with low-speed stirring to form clumps of different sizes spontaneously.

[0085] In particular embodiments, the size of the spheres can range from 50 microns to 500 microns, although small and larger sizes may also be used. In particular embodiments, the sphere size can be between about 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 microns, including any value or range derivable therein. The sphere size may be about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 microns, including any value or range derivable therein. In specific cases, the size of the organoid is generally controlled by the duration of time of culture and the culture media, optionally among other culture parameters.

[0086] In particular embodiments, the organoids comprise a particular range of cell numbers. In specific cases, the number of cells in the organoid are generally controlled by the duration of time of culture and the culture media, optionally among other culture parameters. In specific embodiments, the organoids comprise at least about or no more than about 103, 104, 105, 106, 107, 108, 109, or 1010cells per organoid, including any value or range derivable therein.

[0087] In some embodiments, fibroblast spheroids are generated upon culture in an incubator at about 37 °C, such as for about 24-48 hours. The incubator may or may not be set at about 5% carbon dioxide and greater than about 80% humidity. Stem cell clumps can also form spontaneously at 20-37 °C, although other conditions may be utilized therewith, in some cases.

[0088] In particular embodiments, for formation, the fibroblast cell spheroids may be cultured for 24-48, 24-44, 24-40, 24-36, 24-30, 24-28, 28-48, 28-44, 28-40, 28-36, 28-30, 30- 48, 30-44, 30-40, 30-36, 36-48, 36-44, 36-40, 40-48, 40-44, or 44-48 hours, including any value or range derivable therein, in an incubator of suitable conditions. In specific embodiments, the incubator may be set at a particular temperature, such as at about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 °C, including any value or range derivable therein. A range of temperatures includes about 20-37, 20-35, 20-30, 20-27, 20-25, 20-22, 22-37, 22-35, 22-33, 22-30, 22-27, 22-25, 25-37, 25-35, 25-33, 25-30, 25-27, 27-37, 27-35, 27-33, 27-30, 30- 37, 30-35, 30-33, 33-37, 33-35, or 35-37 °C, including any value or range derivable therein.

[0089] In specific embodiments, the incubator may utilize a certain percentage of a gas, such as carbon dioxide, oxygen, and / or nitrogen. The carbon dioxide may be at 3, 4, 5, 6, 7, or 8%. Ranges of carbon dioxide include about 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5- 7, 5-6, 6-8, 6-7, or 7-8%, including any value or range derivable therein. In specific embodiments, the incubator may be set at a particular percentage of humidity, such as about 65, 70, 75, 80, 85, or 90% humidity, including any value or range derivable therein. Theoxygen in the incubator may be 0.5-25% oxygen, including 0.5-25, 0.5-20, 0.5-15, 0.5-10, 0.5- 5, 0.5-1, 1-25, 1-20, 1-15, 1-10, 1-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or 20-25%, including any value or range derivable therein. The oxygen in the incubator may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, including any value or range derivable therein. The nitrogen in the incubator may be 1-75% nitrogen, including 1-75, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 5-75, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-75, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-75, 20-70, 20-60, 20-50, 20-40, 20-30, 30-75, 30-70, 30-60, 30-50, 30-40, 40-75, 40-70, 40-60, 40-50, 50-75, 50- 70, 50-60, 60-75, 60-70, or 70-75% nitrogen, including any value or range derivable therein. The nitrogen in the incubator may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, or 75% nitrogen, including any value or range derivable therein.

[0090] A range of humidities may be utilized, such as about 65-90, 65-85, 65-80, 65-75, 65-70, 70-90, 70-85, 70-80, 70-75, 75-90, 75-85, 75-80, 80-90, 80-85, or 85-90%, including any value or range derivable therein. Other incubator conditions and culture durations may be present or required.

[0091] In various embodiments, the size of the produced spheroid is able to be controlled at least in part. The desired size may be dictated for one or more purposes, such as a desired size for production purposes, a desired size for transportation purposes, and / or a desired size for a research and / or therapeutic application. In some embodiments, a plurality of organoids to be utilized comprise substantially the same size. In some embodiments, a first plurality of organoids may be combined with a second plurality of organoids of a different size before transportation and / or research and / or therapeutic application; in such cases, the first and second pluralities may have been produced under different conditions, such as different culture parameters, including different durations of culture time. The produced size of the organoids may be dictated by adjusting the concentration of fibroblast cells used to initiate spheroid formation. Such adjusting of the concentration may or may not occur by dilution; by the selection of the base media; by the selection of one or more additives to the media (such as sugar, serum, non-essential amino acids, L-glutamine, a combination thereof, etc.); by selection of the amount of carbon dioxide in the incubator; by the temperature in the incubator; by the duration of time of production of the spheroid; using an outside source of a gas or gas mixtures to feed the incubator; or a combination thereof. The concentration may be increased for certain purposes and decreased for certain purposes. The adjustment of the concentration may increasethe size of the organoid by an approximate certain fold level, such as about 2-fold, 5-fold, 10- fold, 100-fold, 1000-fold, 10,000-fold, etc., including any value or range derivable therein. The adjustment of the concentration may decrease the size of the organoid by an approximate certain fold level, such as about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, etc., including any value or range derivable therein.

[0092] In some embodiments, the size of the organoids can be modified to accommodate varying clinical needs, such as adjusting the concentration of the cells and / or adjusting the duration of time of growth of the organoid. For example, in embodiments wherein a tissue is in need of repair, regeneration and / or reversal of involution, the size of the organoid may be particularly produced, depending on the tissue site in need of the organoids. In some embodiments, the organoid is utilized as a therapeutic delivery agent wherein the therapeutic(s) delivered by the organoid is the fibroblasts themselves and / or one or more agents derived from the fibroblasts. In such cases, the organoid may act as an extended time release mechanism that allows for the release of the therapeutic(s) over a desired period of time. The desired period of time may be when there is a detectable improvement at the site (either by visual or assaying means). The desired period of time may be when there is complete healing at the site (either by visual or assaying means). In some cases, the desired period of time may be the time to be able to initiate or maintain immune modulation as a result of the extended time release. In some cases, the desired period of time may be the time to be able to initiate or maintain activated or inactivated fibroblasts and / or fibroblast-derived material upon delivery and as a result of the extended time release. In some cases, the desired period of time may be the time to initiate or maintain migration and / or proliferation to a targeted tissue or organ for any purpose (including for any purpose, such as repair, regeneration and / or reversal of involution) and as a result of the extended time release. In some cases, the desired period of time may be the time to repair, regenerate and / or reverse involution.

[0093] In some embodiments, the organoids following production are stored under suitable conditions. In at least specific cases, the temperature of the storage environment is about 2 °C to about 37 °C. In specific embodiments, the storage environment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 °C, including any value or range derivable therein. The storage temperature may be in range of suitable temperatures, including at least about 2-37, 2-35, 2-30, 2-25, 2-20, 2-15, 2- 10, 2-5, 5-37, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-37, 10-35, 10-30, 10-25, 10-20, 20-37, 20- 35, 20-30, 20-25, 25-37, 25-35, 25-30, 30-37, 30-35, or 35-37 °C, including any value or range derivable therein.- li

[0094] Once the organoids are generated, they may be stored and / or they may be transported to an intended destination. The organoids may be suspended in a liquid base medium that includes one or more tackifiers to produce a liquid storage matrix. The liquid portion of the liquid storage matrix may comprise any base cell culture medium compatible with fibroblasts, such as one containing basic nutrients and an acid-base balance system; the components may comprise low-glucose medium, such as DMEM low-glucose medium and / or another medium containing 1 mM to 20 mM (e.g., 5 mM) glucose; anywhere from about 0 to 20% human serum; about 0 to 5% non-essential amino acids; and / or about 0 to 5% L-glutamine. The tackifier may be a food-grade additive that can increase the viscosity of the liquid matrix, such as Methylcellulose or any other agent capable of increasing viscosity, and may be at a specific concentration of anywhere from about 0 to 5%. The base medium may contain other materials and / or conditions to facilitate culturing, such as growth factors, sugars, and / or amino acids.

[0095] In embodiments wherein the liquid storage matrix for storage and / or transportation may comprise serum, including human serum, the amount may be about 0-20, 0-15, 0-10, 0-5,1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20% human serum, including any value or range derivable therein. The amount of serum may be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%, including any value or range derivable therein.

[0096] In embodiments wherein the media for storage and / or transportation may comprise non-essential amino acids, the amount may be about 0-5, 0-4, 0-3, 0-2, 0-1, 1-5, 1-4, 1-3, 1-2,2-5, 2-4, 2-3, 3-5, 3-4, or 4-5% non-essential amino acids, including any value or range derivable therein. The amount of non-essential amino acids may be about 0, 1, 2, 3, 4, or 5% non-essential amino acids, including any value or range derivable therein. The non-essential amino acids may include glycine, L-alanine, L-asparagine monohydrate, L-aspartic acid, L- glutamic acid, L-proline, L-serine, L-histidine, Isoleucine, L-lysine hydrochloride, L-serine, L- tryptophan, and / or L-valine.

[0097] In embodiments wherein the liquid storage matrix for storage and / or transportation may comprise about 0 to 5% L-glutamine, the amount may be 0-5, 0-4, 0-3, 0-2, 0-1, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5% L-glutamine, including any value or range derivable therein. The amount of L-glutamine may be about 0, 1, 2, 3, 4, or 5% L-glutamine, including any value or range derivable therein.

[0098] In embodiments wherein the liquid storage matrix for storage and / or transportation may comprise one or more tackifiers, the amount may be 0-5, 0-4, 0-3, 0-2, 0-1, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5% tackifiers, including any value or range derivable therein.In embodiments wherein the media for storage and / or transportation may comprise one or more tackifiers, the amount of tackifier may be about 0, 1, 2, 3, 4, or 5%, including any value or range derivable therein. In cases wherein two or more tackifiers are used, their sum amount may or may not be about 0-5, 0-4, 0-3, 0-2, 0-1, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4- 5%, including about 0, 1, 2, 3, 4, or 5%, including any value or range derivable therein.

[0099] In some embodiments, upon generation the fibroblast spheroids are housed in a suitable container. The range of spheroids per container may vary according to need, but in specific cases the range is 1-10 million cells per milliliter of preservation liquid matrix. In specific embodiments, the packaging and preservation of the fibroblast spheroids are in a sealed container, including a sterile one. In specific embodiments, the container is a plastic container, such as one made from chemically inert materials including polyethylene, polypropylene, and melamine. The container may also be constructed of other materials that would not adversely impact the cells, such as glass, coated metal, or other polymers such as polystyrene, branched polymer hydrogels, and cycle-olefin polymers.

[0100] Embodiments of the present disclosure allow for the spheroids to be transported under ambient conditions, and this may be before or after storage. In specific embodiments, the spheroids are transported in a temperature range from 2-35 °C. The spheroids may be transported in a temperature range of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 °C, including any value or range derivable therein. The spheroids may be transported at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 °C, including any value or range derivable therein. In specific embodiments, the temperature for transport is not more than about 38 °C and / or is not less than 0 °C, although in other embodiments the spheroids are transported cryogenically.

[0101] In specific embodiments, the cell container is not subject to intense energy or light waves, including intense light irradiation, X rays, such as is used at airports, or any intense light source during at least part if not all of transportation. In specific cases, the cell container and / or the incubator during preparation is not subject to light during at least part if not all of transportation.

[0102] The produced organoids may be transported and / or used within about 0-50 days following production. In some cases, the organoids are transported and / or used on the same day as production, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, to 50 days, including any value or range derivable therein. In some embodiments,the organoids are transported and / or used within 1-50, 1-40, 1-30, 1-25, 1-20, 1-10, 1-5, 5-50, 5-40, 5-30, 5-25, 5-20, 5-10, 10-50, 10-40, 10-30, 10-25, 10-20, 20-50, 20-40, 20-30, 20-25, 25-50, 25-40, 25-30, 30-50, 30-40, or 40-50 days, including any value or range derivable therein.

[0103] In particular embodiment, the fibroblast spheroids may be manipulated such that at least some of the cells are dissociated from the organoid, thereby allowing the dissociated fibroblast cells to restore proliferation or to proliferate at a greater rate than in the organoid. In specific embodiments, the organoids are exposed to one or more proteases, mechanical disruption (such as by pipetting up and down), and / or use of sonication for dissociation. In specific cases, the amount of trypsin is about 0.1-0.3% trypsin, such as about 0.25% trypsin. In particular cases, the spheroids are subjected to about 0.25% trypsin and may be placed in a 37 °C incubator to restore proliferation of the dissociated fibroblasts. In addition, the fibroblasts can be directly plated back into the vessel it was transported in and cultured in normothermia.

[0104] In particular embodiments, a plurality of fibroblasts can be released from the spheroids. The rate of release can vary based on size of the spheroids, content of spheroid, how the spheroids were activated, and / or the environment in which it was administered. In particular embodiments, a plurality of fibroblasts can be released from the spheroids and migrate to a desired site (such as a specific organ or tissue). The ability of the released fibroblasts to migrate to a desired site may be because of one or more natural characteristics of the released fibroblasts or because of one or more acquired characteristics of the released fibroblasts, such as characteristics imparted to the fibroblasts by the hand of man. In specific embodiments, the released fibroblasts migrate to a desired site because the cells have become activated by activated or engineered surface markers, nucleic acid modification, and / or expression or excretion of one or more chemokines, one or more cytokines, exosomes, chimeric antigen receptors, and / or one or more growth factors. In particular embodiments, a plurality of fibroblasts can be released from the spheroids and migrate to a desired site and grow adherently from the spheroids. In specific embodiments, when the spheroids bind to the desired surface, the cells then migrate out of the spheroids onto that desired area and may proliferate. Furthermore, in some embodiments, the fibroblast spheroids can separate from the preservation liquid matrix and directly applied to transplantation site for tissue regeneration or immunotherapy.

[0105] In some embodiments, the size of the organoids can be modified to accommodate varying clinical needs, such as adjusting the concentration of the cells and / or adjusting the duration of time of growth of the organoid. For example, in embodiments wherein a tissue isin need of repair, regeneration and / or reversal of involution, the size of the organoid may be particular, depending on the tissue site in need of the organoids. In some embodiments, a specific size of organoid is desired to be able to be injected from a needle. In some embodiments, specific size of organoid is desired to be able to be infused to a specific site in which the vein / artery size.

[0106] In some embodiments, the organoid is utilized as a therapeutic delivery agent wherein the therapeutic(s) delivered by the organoid is the fibroblasts themselves and / or one or more agents derived from the fibroblasts. In such cases, the organoid may act as an extended time release mechanism that allows for the release of the therapeutic(s) over a desired period of time. The desired period of time may be when there is a detectable improvement at the site (either by visual or assaying means). The desired period of time may be when there is complete healing at the site (either by visual or assaying means). In some cases, the desired period of time may be the time to be able to initiate or maintain immune modulation as a result of the extended time release. In some cases, the desired period of time may be the time to be able to initiate or maintain activated or inactivated fibroblasts and / or fibroblast-derived material upon delivery and as a result of the extended time release. In some cases, the desired period of time may be the time to initiate or maintain migration and / or proliferation to a targeted tissue or organ for any purpose (including for any purpose, such as repair, regeneration and / or reversal of involution) and as a result of the extended time release. In some cases, the desired period of time may be the time to repair, regenerate and / or reverse involution at a desired site.

[0107] In particular embodiments, one can modulate the size of the fibroblast spheroids to adjust the extent and duration of the extended-release function of single fibroblast cells from the organoids and or fibroblast-derived materials in vivo. Such modulation may encompass producing larger-sized organoids for longer extended release of the cells and / or fibroblast- derived materials vs. producing smaller-sized organoids for extended release of the cells and / or fibroblast-derived materials that are not needed for so long. For example, for organ involution, in specific embodiments one may produce organoids that are larger in size than for localized tissue repair.

[0108] In embodiments of the disclosure, one may utilize the organoid fibroblasts as a method of “extended time release” of the fibroblast cells and / or fibroblast cell-derived or excreted materials. Such agents may be used for any suitable purpose, including for the purpose of immune modulation, chronic disease treatment, tissue repair, tissue regeneration, targeting of specific tissue for migration, and / or proliferation for subsequent growth, and / or differentiation.

[0109] In specific embodiments, the fibroblasts from the organoids and / or fibroblast- derived materials from the organoids may be released slowly over a period of time from the spheroid to elicit an immune modulation response or other biological responses that is therapeutic for the treatment of one or more ulcerative conditions, such as aphthous ulcers. In specific embodiments, the organoids may be used in treatment of cancer-related ulcers of any kind, such as ulcers caused by chemotherapy, radiotherapy, or immunotherapy.

[0110] In particular embodiments, the fibroblast organoids are administered to an individual in need thereof by any suitable administration, including one that is selected for the application being treated. In specific embodiments, the fibroblast organoids are dissociated before administration. In specific embodiments, fibroblast-derived factors from the fibroblast’s organoids are isolated and used for administration. Such administration may be local or systemic and may include injection, infusion, spray, and / or inclusion in a 3D matrix. In specific embodiments, the fibroblast organoids, dissociated fibroblast organoids, and / or fibroblast- derived factors from the fibroblasts organoids are comprised in a carrier for buccal delivery, such as but not limited to, mouthwash, dentifrice, buccal tablet, lollipop-like structure, chewing gum, oral spray, lozenge, toothpaste, or sublingual orally-administered formulation. In specific embodiments, there is one administration to the individual or multiple administrations. In cases wherein there are multiple administrations, the duration in time between the administrations may be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or within 1, 2, 3, 4, 5, 6, or 7 days, or within 1, 2, 3, or 4 weeks, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or within 1, 2, 3, 4, 5, or more years between administrations, including any value or range derivable therein. In specific embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations may occur within a 24 hour period.[OHl] In specific embodiments, the organoids, dissociated fibroblast organoids, and / or fibroblast organoid derived factors are delivered to or onto a damaged tissue; in specific embodiments thereafter, the fibroblasts migrate and / or proliferate to a desired site, such as to regenerate the damaged tissue through differentiation, immune modulation, and / or stem cell recruitment.

[0112] In certain embodiments, the organoid fibroblasts have been activated, such as to express one or more surface molecules that target a specific tissue or organ for migration and proliferation for tissue repair and / or eliciting an immune response at the target site. The fibroblasts may be activated by any suitable means, such as through one or more chemical agents (including small molecules), RNA, micro RNA, RNAi , DNA, viral nucleic acid, and / or exosomes. Specific examples include 6SM, p53 peptide, I09 / RG7388, NUT / nutlin-3a, IQK,41P, 2FS, TX6 / TX64014, Estradiol, 4-Hydroxytamoxifen, Bazedoxifene, AZD9496, Testosterone, Bicalutamide, PT2399, ZTD, VH298, T28, OWC, 76Z, JQ1, or a combination thereof. In specific embodiments, the organoid fibroblasts have been genetically modified to comprise a chimeric antigen receptor by methods known in the art (see e.g., U.S. Patent Application 17 / 271,529, which is incorporated by reference herein in its entirety).

[0113] In some embodiments, fibroblast organoids are cultured with other cells (e.g., platelets, neutrophils, monocytes, macrophages (e.g., Ml, M2, M2a), dendritic cells (e.g., Langerhans cells), T cells (e.g., aP T cells, y5 T cells, T helper cells, regulatory T cells, killer T cells), endothelial cells, pericytes, hematopoietic progenitor cells, epidermal cells, epidermal stem cells, smooth muscle cells, keratinocytes, lymphocytes, mast cells, NK cells, adipose stromal cells, other fibroblasts, or a combination thereof). In some embodiments, cells or derived factors from organoid co-cultures are used in the methods described herein.E) Activated Fibroblasts

[0114] Embodiments of the disclosure include the use of fibroblasts or fibroblast-derived materials, fibroblasts activated with one or more agents, and / or fibroblasts (including myofibroblasts or dermal fibroblasts) plus one or more types of immune cells, keratinocytes, and / or basal epithelial cells and / or derivatives thereof into, on top of, and / or adjacent to the wound for the purpose of initiating or maintaining wound healing.

[0115] In particular embodiments, fibroblasts or other cells encompassed herein become activated following exposure to one or more conditions and / or agents. Specific agents that may modify the fibroblasts include one or more of nucleic acids, cytokines, chemokines, growth factors, and / or exosomes prior to and / or during their use. The fibroblast cells may be activated, such as having activated or engineered surface markers, nucleic acid modification, and / or expression or excretion of one or more chemokines, one or more cytokines, exosomes, and / or one or more growth factors. In specific embodiments, the fibroblasts are activated by exposing them to other cells (e.g., platelets, neutrophils, monocytes, macrophages (e.g., Ml, M2, M2a), dendritic cells (e.g., Langerhans cells), T cells (e.g., aP T cells, y5 T cells, T helper cells, regulatory T cells, killer T cells), endothelial cells, pericytes, hematopoietic progenitor cells, epidermal cells, epidermal stem cells, smooth muscle cells, lymphocytes, mast cells, NK cells, adipose stromal cells, immune cells, keratinocytes, epithelial cells, other fibroblasts, or a combination thereof). The cells may be activated by one or more chemical agents, RNA, micro RNA, RNAi , DNA, viral nucleic acid, and / or exosomes. In specific embodiments, the cytokineis selected from the group consisting of IFN-gamma, TNF-alpha, interleukin (IL)-l, IL-6, IL- 7, IL-8, IL-12, IL-15, IL-17, IL-33, and a combination thereof. In specific embodiments, the growth factor is selected from the group consisting of FGF-1, VEGF, and a combination thereof. In specific embodiments, the cells are activated following exposure to human platelet-rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, human serum, serum replacement, or a combination thereof. In specific embodiments, the cells are activated following exposure to hypoxia. The hypoxia may be 0.1 %- 10%, 0.1%-5%, 0.1%-2.5%, or 0.1%- 1% oxygen, for example. In specific embodiments, the hypoxia occurs for a period of time that is at least or no more than between about 30 minutes to about 3 days, although in some cases, it may be less than 30 minutes or greater than 3 days. The fibroblasts may be exposed to one or more agents prior to and / or during exposure to hypoxia, carbon monoxide, or a combination thereof.F) Production of Fibroblast-Derived Factors

[0116] In some embodiments, fibroblasts and / or fibroblast-derived materials (including fibroblast cells; fibroblast-like cells; fibroblast-derived lysate; and / or extracellular vesicles including exosomes, microvesicles, apoptotic bodies or any other fragments or biologic components of fibroblast cells) produce therapeutic factors that can initiate, maintain, and accelerate the wound healing process in aphthous ulcers.

[0117] During cell culture, fibroblast or fibroblast-like cells naturally secrete various factors. These factors include growth factors (e.g., fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, keratinocyte growth factor, TGF-P), cytokines (e.g., interleukin-6, transforming growth factor-beta), extracellular matrix components (e.g., collagen, fibronectin, integrins), and other bioactive molecules (e.g., matrix metalloproteases, WNTs (8, 13)). Other factors include IFN- y, EGFR, IL- 10, IL-1 a, and / or IL-6.

[0118] In some embodiments, introduction of fibroblast-derived factors into, on top of, and / or adjacent to the wound, e.g., an aphthous ulcer, results in partial or full healing, for example results in (1) tissue differentiation; (2) cell recruitment and differentiation of stems cells at the wound site; (3) expansion of already locally present keratocytes, vasal epithelial cells, myofibroblasts, endothelial cells, immune cells, and / or dermal fibroblasts; and / or (4) attraction of other cells or biologic processes to aid in the healing, all for the purpose of initiating or maintaining wound healing. In some embodiments, the healing by any mechanismoccurs at a faster rate than the healing of the wound in the absence of the disclosed methods or compositions.

[0119] In some embodiments, fibroblast-derived factors may be obtained from fibroblast conditioned media. Conditioned media contains at least one or more of metabolites, growth factors, and / or extracellular matrix proteins secreted into the medium by the cultured cells. Examples may include metabolites such as glucose, amino acids, and nucleosides; growth factors such as interleukins, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF); and matrix proteins such as collagen, fibronectin, and various proteoglycans. Fibroblasts are capable of producing and releasing into the culture media various immune modulators including peptide growth factors, cytokines, chemokines and inflammatory mediators.

[0120] In cases when fibroblasts are cultured under hypoxic conditions, the fibroblasts may secrete factors selected from the group consisting of: a) monocyte chemoattractant protein 1 (MCP-1); b) macrophage inflammatory protein 1 beta (MIP-ip); c) IL-6; d) IL-8; e) granulocyte chemotactic protein 2 (GCP-2); f) HGF; g) keratinocyte growth factor (KGF); h) FGF; i) heparin binding EGF-like growth factor (HB-EGF); j) brain-derived neurotrophic factor (BDNF); k) thyroid peroxidase (TPO); 1) RANTES (also known as CC motif chemokine ligand 5); m) TIMP metallopeptidase inhibitor 1 (TIMP1); and n) a combination thereof.G) Chimeric Antigen Receptors

[0121] In particular aspects, chimeric antigen receptors (CARs) are utilized in fibroblast cells for a therapeutic purpose and are comprised of multiple components that are not similarly grouped in a natural setting. The CARs are synthetic and made by the hand of man. Included in the scope of the disclosure are functional portions of the inventive CARs described herein. In one embodiment the CAR is utilized to activate fibroblast cells, induce expression or release of therapeutic factors, target fibroblasts cells to an ulcer, or a combination thereof. Engineering of fibroblasts to comprise a CAR can be achieved by methods know in the art (see e.g., U.S. Patent Application 17 / 271,529, which is incorporated herein by reference in its entirety).

[0122] In specific embodiments, the intracellular domain of the CAR comprises at least a signal transduction domain, e.g., CD3 zeta chain. In addition to this, or as an alternative, the CAR may comprise one or more intracellular domains, and the intracellular domains may or may not have the activity of being costimulatory domains. When the intracellular domain comprises a costimulatory domain, the costimulatory domain may be any one or more of, forexample, members of the TNFR superfamily, CD40L, CD28, CD137 (4-1BB), CD134 (0X40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1 la / CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. As one example, the intracellular domain may comprise an activator of one or more molecular pathways that endows an immune stimulatory phenotype to the fibroblast, such as an intracellular domain of the TLR-4 protein. In some embodiments, the fibroblast may further comprise at least one shRNA that generates at least one siRNA capable of inhibiting expression of HLA I and / or HLA II (including the HLA genes that are non-polymorphic). The fibroblasts are engineered in a manner such that binding of the CAR to its cognate ligand results in transduction of a signal stimulating activation of the fibroblast and release of one or more factors. In some embodiments, the CAR-expressing fibroblast secretes TRAIL, TNF-alpha, or TNF-beta. In some embodiments, such CAR- expressing fibroblasts secrete one or more immune stimulatory cytokines, such as IL- 12, IL- 15 or IL-21. In some embodiments, the CAR-expressing fibroblast secretes growth factors, angiogenic factors, and / or antibacterial factors. In some embodiments, the CAR-expressing fibroblast secretes defensin.

[0123] The CARs encompassed herein may be synthesized as single polypeptide chains and may comprise at least one antigen-specific targeting region, optionally an extracellular spacer domain, a transmembrane domain, and one or more intracellular signaling domains. In one embodiment, the antigen-specific targeting region(s) are at the N-terminus, and if the CAR is bispecific they may be arranged in tandem and may be separated by a linker peptide. The antigen-specific targeting region optionally may be linked to an extracellular spacer domain which is linked to the transmembrane domain. The transmembrane domain may be linked to an intracellular signaling domain that may be at the C-terminus. Polynucleotides encoding these polypeptides may further comprise an N-terminal signal sequence that directs the CAR to the cell surface as a type I transmembrane protein. The antigen-specific targeting region may be extracellular-facing and the intracellular signaling domain may be cytoplasmic.

[0124] In cases where the CAR is a bispecific CAR, the antigens targeted by the bispecific CAR may be antigens on a single cell or antigens that are expressed on separate cells. The antigen(s) targeted by the CAR are antigen(s) that are either directly or indirectly involved in the ulcer. In a bispecific CAR, at least two different antigen-specific antibodies or fragments thereof or derivatives thereof may be cloned into the antigen-specific targeting region. The antibodies may be specific for any, but at least two, distinct antigens of choice. The antibody specific to the antigen may be the Fab fragment of the antibody or the single chain variable fragment (scFv) of the antibody.

[0125] In one embodiment, CAR-transfected fibroblasts are generated with one or more CARs comprising an antigen binding domain that is capable of binding to an antigen, such as an injury associated antigen, e.g., tissue factor, calreticulin, and / or extracellular vimentin.

[0126] In one embodiment, the CAR drives the expression of one or more target genes. The target gene may be an endogenous gene or a heterologous gene. The heterologous gene may be provided as a gene cassette comprising a promoter that is activated by the activity of the CAR. Alternatively, the heterologous gene heterologous gene may comprise a drug inducible promoter. A heterologous gene may be provided to the fibroblast by any suitable method known in the art, e.g., transfection. The heterologous gene may be provided to the fibroblast before, concurrently, or after a fibroblast is provided with a CAR. The target gene may be a growth factor, cytokine, angiogenic factor, antibacterial factor, or a combination thereof.

[0127] The definition of "functional portion" when used in reference to a particular CAR refers to any part or fragment of the CAR of the disclosure, said part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease or condition, to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the parent CAR. The functional portion can comprise additional amino acid(s) at the amino or carboxy terminus of the portion, or at both termini, said additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect ulcer site, or treat an ulcer. A functional variant can, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR. Amino acid substitutions of the CARs are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acidthat has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0128] The CAR can comprise, consist of, or consist essentially of the specified amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not materially change the biological activity of the functional variant. The CARs of the disclosure retain their biological activity, e.g., the ability to specifically bind to antigen, target to an ulcer site, or treat an ulcer in an individual, etc. For example, the CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0129] CARs of embodiments of the disclosure (including functional portions and functional variants of the disclosure) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, alpha-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, beta-phenylserine, beta-hydroxyphenylalanine, phenylglycine, alpha-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2,3,4- tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide’ N'-benzy’-N'-methyl-lysine’ N’,N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, alphaaminocyclopentane carboxylic acid, alpha-aminocyclohexane carboxylic acid, alphaaminocycloheptane carboxylic acid, alpha-(2-amino-2-norbornane)-carboxylic acid, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine, and alpha-tert-butylglycine. The CARs of embodiments of the disclosure (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated,esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted, dimerized, polymerized, or conjugated.

[0130] The CARs of embodiments of the disclosure (including functional portions and functional variants thereof) can be obtained or generated by methods known in the art. The CARs may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Pat. No. 5,449,752, all of which are incorporated herein by reference in their entirety. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994, all of which are incorporated herein by reference in their entirety. Further, some of the CARs of the invention (including functional portions and functional variants thereof) can be isolated and / or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies, such as Synpep (Dublin, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems (San Diego, Calif.). In this respect, the CARs can be synthetic, recombinant, isolated, and / or purified.

[0131] In some embodiments, activation of a CAR induces the expression, and / or secretion of factors that promote ulcer healing, for example, but not limited to collagens, glycosaminoglycans, hyaluronic acid, elastin, laminins, tenascins, fibronectin, endothelin, catecholamines, epinephrine, norepinephrine, prostaglandins, bradykinin, fibrinopeptide, serotonin, histamine, thromboxane A2, myeloperoxidase, azurocidin, lysozyme, bacterial permeability increasing protein, cathepsin G, elastase, protease 3, human cationic antimicrobial protein 18, lactoferrin, matrix metalloprotease 8, collagenase 2, chymase, tryptase, matrix metalloproteases, tissue inhibitor of metalloproteinases- 1, -2, -3, angiopoietins, FGF, KGF, VEGF, PDGF, TGF, EGF, IGF, TNF-alpha, IL-6, IL 1 -beta, INF-alpha, INF-beta, IL-2, IL-4, IL-8, IL- 10, IL-7, hyaluronan, osteopontin, periostin, vitronectin, angiotensin, CCN2, Cx43,CXCL8, CXCL1, CXCL12, CXCL2, RANTES, MCP, MIPs, lymphotactin, CCL2, CXCL10, fibrinogen, von Willebrand factor, collagen, fibrin, biglycan, decorin, versican, heparan sulfate, tenascin C, uric acid, S100 protein, ATP, F-actin, cyclophilin A, histones, HMGB1, IL-1 alpha, IL-33, SAP130, DNA, RNA, mtDNA, formyl peptide, mROS, calreticulin, defensins, cathelicidin, eosinophil-derived neurotoxin, granulysin, syndecans, glypicans, mast cell protease-4 or -5, CCN1, stromal derived factor-1 (SDF-1 / CXCL12), monocyte chemoattractant protein-1 (MCP-1), Tie-2, SKINTs, CD100, ICAM-1, VCAM-1, P-selectin, and / or E-selectin. i) Antigen-Specific Targeting Region

[0132] In particular embodiments the CAR molecule comprises at least one antigenspecific targeting region that targets an antigen, such as an antigen present at the site of an ulcer. The antigen-specific targeting region may target a cell antigen, extracellular protein antigen, injury associated antigen, or a pathogen antigen, for example.

[0133] In specific embodiments, the antigen-specific targeting region comprises an antibody, or antigen binding portion thereof. The antibody can be any type of immunoglobulin that is known in the art. For instance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally- occurring antibody, e.g., an antibody isolated and / or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be a genetically-engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody can be in monomeric or polymeric form. Also, the antibody can have any level of affinity or avidity for the target antigen. In specific embodiments, antibodies that bind to a CAR encompassed by the disclosure are utilized to render the CAR-expressing fibroblasts ineffective, such as when they are no longer needed.

[0134] In one embodiment, an antigen-specific targeting region comprises the full-length IgG heavy chain (specific for the target antigen) having the VH, CHI, hinge, and the CH2 and CH3 (Fc) Ig domains, if the VH domain alone is sufficient to confer antigen-specificity (“single-domain antibodies”). The full length IgG heavy chain may be linked to the costimulatory domain and the intracellular signaling domain via the appropriate transmembrane domain. If both the VH and the VL domains are necessary to generate a fully active antigenspecific targeting region, the VH-containing CAR and the full-length lambda light chain (IgL) may both be introduced into the fibroblast cells to generate an active antigen-specific targetingregion. In an embodiment, an extracellular spacer domain may be linked between the antigenspecific binding domain and the transmembrane domain.

[0135] In a certain embodiment, an antigen-specific targeting region of the CAR comprises a single chain antibody variable fragment (scFv) specific for a target antigen (and bispecific CARs comprise two scFvs specific for non-identical target antigens). scFvs, in which the C- terminus of one variable domain (VH or VL) is tethered to the N-terminus of the other (VL or VH, respectively) via a polypeptide linker, have been developed without significantly disrupting antigen binding or specificity of the binding, e.g., Chaudhary et al., A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin. 1990 Proc. Natl. Acad. Sci., 87:9491; Bedzyk et al. Immunological and structural characterization of a high affinity anti -fluorescein single-chain antibody. 1990 J. Biol. Chem., 265: 18615, all of which are incorporated herein by reference in their entirety. The linker connects the N-terminus of the VH with the C-terminus of VL or the C-terminus of VH with the N-terminus of VL. These scFvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. The scFvs are arranged linked to the intracellular signaling domain via a transmembrane domain. In an embodiment, an extracellular spacer domain may be linked between the antigen-specific binding region and the transmembrane domain.

[0136] In another aspect, a scFv fragment may be fused to all or a portion of the constant domains of the heavy chain. The resulting antigen-specific targeting region, specific for one or at least two different antigens, is joined to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an embodiment, an extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

[0137] In a further embodiment, an antigen-specific targeting region of the CAR comprises a divalent (or bivalent) single-chain variable fragment (di-scFv, bi-scFv). In bispecific CARs comprising di-scFvs, two scFvs specific for each antigen are linked together by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs, see e.g., Xiong, Cheng- Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). “Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding.” Protein Engineering Design and Selection 19 (8): 359-367; Kufer, Peter; Lutterbuse, Ralf; Baeuerle, Patrick A. (2004). “A revival of bispecific antibodies.” Trends in Biotechnology 22 (5): 238-244, both of which are incorporated herein by reference in their entirety. CARs comprising at least two antigen-specific targeting regions would express two scFvs specific for each of the two antigens. The resulting antigen-specific targeting region,specific for at least two different antigens, is joined to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an embodiment, an extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

[0138] In an additional embodiment, an antigen-specific targeting region of the CAR comprises a diabody. In a diabody, the scFvs are created with linker peptides that are too short for the two variable regions to fold together, driving the scFvs to dimerize. Still shorter linkers (one or two amino acids) lead to the formation of trimers, the so-called triabodies or tribodies. Tetrabodies may also be used.

[0139] To generate bispecific CARs, two or more individual antigen-specific targeting regions are connected to each other, either covalently or noncovalently, on a single protein molecule. An oligopeptide or polypeptide linker, an Fc hinge or membrane hinge region may be used to connect these domains to each other. In bispecific CARs, they may comprise two or more of the different antigen-specific targeting regions connected together in different combinations. For example, two or more antigen- specific targeting regions containing immunoglobulin sequences (e.g. scFvs and / or single-domain antibodies) may be linked to each other.

[0140] Targets of antigen-specific targeting regions of CARs may be of any kind. In some embodiments, the antigen-specific targeting region of the CAR targets antigens specific for injury, inflammation, infection, cancer, inflammatory disease, neuronal-disorders, diabetes, cardiovascular disease, infectious diseases or a combination thereof. Examples of antigens that may be targeted by the CARs include but are not limited to injury associated antigen, e.g., tissue factor, calreticulin, and / or extracellular vimentin, antigens expressed on B-cells, antigens expressed on carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and / or inflammatory diseases. The CARs of the disclosure may be capable of redirecting the effector function of the expressing-cells to the target antigen(s).

[0141] In particular embodiments, the CAR(s) of the CAR-expressing fibroblasts have affinity for a particular injury associated antigen. In an exemplary embodiment, the genetically engineered cells of the disclosure express a CAR that is specific for tissue factor, calreticulin, extracellular vimentin antigen(s), TNF-alpha, mast cell protease-4 or -5, CCN1, stromal derived factor-1 (SDF-1 / CXCL12), monocyte chemoattractant protein-1 (MCP-1), Tie-2, SKINTs, CD100, ICAM-1, VCAM-1, P-selectin, E-selectin, and / or CXCL10.

[0142] Embodiments of the disclosure include fibroblast cells that express a chimeric antigen receptor (CAR) comprising: a) at least one extracellular antigen-specific targeting region; b) at least one transmembrane domain; and c) at least one intracellular signaling domain. In specific embodiments, the antigen-specific targeting region targets an injury associated antigen.

[0143] Antigens that may be targeted by the CARs of the disclosure include but are not limited to any one or more of von Willebrand factor, tissue factor, calreticulin, extracellular vimentin antigen(s), damage-associated molecular patterns (DAMPs; e.g., biglycan, decorin, versican, hyaluronan, heparan sulfate, tenascin C, uric acid, SI 00 protein, ATP, F-actin, cyclophilin A, histones, HMGB1, IL- 1 alpha, IL-33, SAP 130, DNA, RNA, mtDNA, formyl peptide, mROS, calreticulin, defensins, cathelici din, eosinophil-derived neurotoxin, granulysin, syndecans, and / or glypicans), Complement 3a (C3a), C5a and so forth. Other injury specific antigens will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0144] In some embodiments, the bispecific chimeric antigen receptors target and bind at least two different antigens. Examples of pairings of at least two antigens bound by the bispecific CARs of the disclosure include but are not limited to any combination with tissue factor, calreticulin, extracellular vimentin antigen(s), TNF-alpha, mast cell protease-4 or -5, CCN1, stromal derived factor-1 (SDF-1 / CXCL12), monocyte chemoattractant protein-1 (MCP-1), Tie-2, SKINTs, CD 100, ICAM-1, VCAM-1, P-selectin, E-selectin, CXCL10, von Willebrand factor, tissue factor, calreticulin, and / or extracellular vimentin antigen(s), damage- associated molecular patterns (DAMPs), Complement 3a (C3a), C5a, and / or any other antigen disclosed herein. Other pairings of antigens specific for ulcer or wound targeting will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.

[0145] Antigens specific for inflammation that may be targeted by the CARs of the disclosure include but are not limited to any one or more of AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin a4p7, Lama glama, LFA-1 (CD1 la), MED 1-528, myostatin, OX-40, rhuMAb P7, scleroscin, SOST, TGF beta 1, TNF-a or VEGF-A. Other antigens specific for inflammation diseases will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0146] Antigens specific for infection that may be targeted by the CARs of the disclosure include but are not limited to any one or more of anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1 , Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus and TNF-a. Other antigens specific for infection will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.

[0147] Yet further targets of the CARs of the disclosure will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. ii) Transmembrane Domain

[0148] The CARs of the disclosure may also comprise a transmembrane domain. The transmembrane domain may comprise the transmembrane sequence from any protein that has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of the disclosure may also comprise an artificial hydrophobic sequence. The transmembrane domains of the CARs of the disclosure may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.

[0149] In particular embodiments, the CAR comprises a transmembrane domain selected from the group consisting of CD3-zeta, CD28, CD8-alpha, CD4, or a combination thereof, for example. iii) Intracellular Signaling Domain

[0150] The intracellular signaling domain may be of any kind, but in specific embodiments it is linked to an activator of a molecular pathway that endows the CAR-stimulated fibroblast with an immune stimulatory phenotype, a regenerative phenotype, or a chemotactic phenotype. For example, the CAR-stimulated fibroblast may have enhanced ability to stimulate a mixed immune cell reaction (mixing immune cells with the fibroblasts and seeing if the immune cells proliferate or produce cytokines in response) as compared to a naive fibroblast. For example, the immune stimulatory phenotype may be an enhanced ability to inhibit infection, clear cell debris, or promote recruitment of immune cells to the ulcer as compared to a naive fibroblast or a fibroblast that does not express a CAR. The regenerative phenotype may be, for example, increased expression regenerative factors, e.g., growth factors, or cytokines, increasedproliferation, or a combination thereof. The chemotactic phenotype may be, for example, an increased recruitment of immune cells, stem cells, epithelial cells, vascular cells, nerve cells, any other cell involved in the healing process, or combination thereof.

[0151] In particular embodiments, the intracellular signaling domain is linked to one or more activators of a molecular pathway that endows an immune stimulatory phenotype and in specific embodiments the activator of molecular pathways that endows an immune stimulatory phenotype is an intracellular domain of the TLR-4 protein. In particular aspects, the activator of molecular pathways that endow an immune stimulatory phenotype is the functional portion of the TLR-4 protein that interacts with MyD88 at a sufficient affinity to trigger MyD88 signal transduction. In specific cases, the activator is the functional portion of the TLR-4 protein that interacts with TRAM and MAL at a sufficient affinity (for example, as seen with phosphorylation assays) to trigger TLR4 signal transduction.

[0152] In some embodiments, the intracellular signaling domain comprises a protein domain that activates cytokines, including for example, IL-12, IL-7, IL-15, and / or IL-21, or stimulates an immune cell, e.g., a T cell. Examples include JAK (Janus activating kinase) and ITAMs (Immune tyrosine activating motifs) protein domains.

[0153] In specific embodiments, an intracellular signaling domain transduces the effector function signal and directs the cell to perform its specialized function. Examples of intracellular signaling domains include, but are not limited to, C, chain of the T-cell receptor or any of its homologs, e.g., r] chain, FcsRly and P chains, MB1 (Iga) chain, B29 (Ig ) chain, etc.), CD3 polypeptides (A, 5 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.H) Cell Engineering

[0154] Certain embodiments relate to cells comprising polypeptides or nucleic acids of the disclosure.

[0155] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least somecases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific embodiments, a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0156] Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). i) Transduction

[0157] As used herein the term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell by means of a virus or viral vector. In some embodiments, this transduction is done via a viral vector.

[0158] In certain aspects, the term “viral vector” intends a recombinant vector that retains the ability to infect and transduce nondividing and / or slowly-dividing cells and may integrate into the target cell’s genome. In some aspects, the vector is derived from or based on a wildtype virus. In some aspects, the vector is derived from or based on a wild-type lentivirus. Examples of such include without limitation, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (Hy). Alternatively, it is contemplated that other retrovirus can be used as a basis for a vector backbone such murine leukemia virus (MLV). It will be evident that a viral vector according to the disclosure need not be confined to the components of a particular virus. The viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Viral vector components may be manipulated to obtain desired characteristics, such as target cell specificity.

[0159] The recombinant viral vectors of this disclosure may be derived from primates and non-primates. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype “slow virus” visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior artrecombinant lenti-viral vectors are known in the art, e.g., see U.S. Pat. Nos. 6,924,123; 7,056,699; 7,419,829 and 7,442,551, incorporated herein by reference. ii) Transfection

[0160] As used herein, the term “transfection” is defined as the introduction of an extracellular nucleic acid into a host cell by any means known in the art, including calcium phosphate co-precipitation, viral transduction, liposome fusion, microinjection, microparticle bombardment, electroporation, etc. The terms “uptake of nucleic acid by a host cell”, “taking up of nucleic acid by a host cell”, “uptake of particles comprising nucleic acid by a host cell”, and “taking up of particles comprising nucleic acid by a host cell” denote any process wherein an extracellular nucleic acid, with or without accompanying material, enters a host cell.

[0161] A variety of methods are known in the art and suitable for transfection of nucleic acid into a cell. The polynucleotides of the present invention may be formulated, using the methods described herein. The formulations may contain polynucleotides which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and / or a sustained-release delivery depot.

[0162] The formulated polynucleotides may be delivered to the cell using routes of administration known in the art and described herein. Examples of typical methods include, but are not limited to, naked delivery, lipidoid mediate transfer, liposome-, lipoplexes, and / or lipid nanoparticle-mediated transfer, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.IV. Extraction and / or Concentration

[0163] The therapeutic factors produced by the cultured cells are extracted and / or concentrated using appropriate techniques, such as centrifugation, filtration, or chromatography. This step ensures the isolation and purification of the bioactive molecules from the cell culture supernatant or lysate.

[0164] Certain embodiments include production of one or more factors from fibroblast cultures through the use of filters that separate compositions based on electrical charge, size and / or ability to elute from an adsorbent. Numerous techniques are known in the art forpurification of cell-derived factors and concentration of agents. For some particular uses fibroblast derived compounds are sufficient for use as culture supernatants of the cells in media. Currently media useful for this purpose include at least Roswell Park Memorial Institute (RPMI-1640), Dublecco's Modified Essential Media (DMEM), Eagle's Modified Essential Media (EMEM), Optimem, and Iscove's Media.

[0165] In some embodiments, a sufficient amount of fibroblast lysate may be generated. Generation of lysate from cells is routine in the art.A) Concentration

[0166] In certain embodiment, fibroblast-derived factors capable of promoting aphthous ulcer healing, are derived from cell cultures and may comprise exosomes, lysate, conditioned media, metabolites, vesicles, apoptotic bodies, any other secreted factor, or a combination thereof. In such an embodiment, the factors may be concentrated by filtering / desalting means known in the art including use of ultracentrifugal membrane filters (e.g., Amicon® filters) with specific molecular weight cut-offs, said cut-offs may select for molecular weights higher than 1 kDa to 50 kDa. In some embodiments, supernatant, or other compositions containing fibroblast-derived factors, may be concentrated using means known in the art such as solid phase extraction using C18 cartridges (e.g., Mini-Spe-ed Cl 8-14%, S.P.E. Limited, Concord ON). The cartridges may be prepared by washing with methanol followed by deionized- distilled water. Up to about 100 mL of fibroblast conditioned media supernatant may be passed through each of these specific cartridges before elution, and it is understood by one of skill in the art that larger or smaller cartridges may be used. After washing the cartridges, material adsorbed is eluted, such as with 3 mL methanol, evaporated under a stream of nitrogen, redissolved in a small volume of methanol, and optionally stored at 4 °C. Before testing the eluate for activity in vitro and / or in vivo, the methanol is evaporated under nitrogen and replaced by culture medium. The Cl 8 cartridges may be used to adsorb small hydrophobic molecules from the fibroblast conditioned supernatant and allows for the elimination of salts and other polar contaminants.

[0167] It may, however be desired to use other adsorption means in order to purify certain factors. The concentrated supernatant, or other compositions containing fibroblast-derived factors, may be assessed directly for biological activities useful for the practice of this disclosure, or may be further purified. Further purification may be performed using, for example, gel filtration using a Bio-Gel P-2 column with a nominal exclusion limit of 1800 Da(e.g., Bio-Rad, Richmond Calif.). Said column may be washed and pre-swelled in 20 mM Tris- HC1 buffer, pH 7.2 (Sigma) and degassed by gentle swirling under vacuum. Bio-Gel P-2 material be packed into a 1.5x54 cm glass column and equilibrated with 3 column volumes of the same buffer. Fibroblast cell supernatant concentrates, for example, may be extracted by Cl 8 cartridge and dissolved in 0.5 ml of 20 mM Tris buffer, pH 7.2 and run through the column. Fractions may be collected from the column and analyzed for biological activity. Other purification, fractionation, and identification means are known to one skilled in the art and include anionic exchange chromatography, gas chromatography, high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. Supernatant active fractions may be used in the compositions and / or methods disclosed herein.B) Exosome Purification

[0168] Embodiments of the disclosure include one or more compositions and methods of use thereof for the treatment of aphthous ulcers, and in specific embodiments the composition or methods of use may comprise exosomes from cultured fibroblasts. In some embodiments the exosomes are generated by a process comprising the steps of: a) obtaining one or more fibroblast cells; b) culturing said fibroblast cells under conditions to allow for production of exosomes into culture media within which said fibroblast cell is cultured; c) obtaining exosomes from the culture media. In particular embodiments the exosomes are administered to an individual in need of treatment. The fibroblasts from which the exosomes are derived may be derived from a biopsy, wherein a donor providing the biopsy is either the individual to be treated (autologous) or the donor is different from the individual to be treated (allogeneic). In specific cases the fibroblasts are cultured in a media allowing for fibroblast proliferation, and the media allowing for fibroblast proliferation may comprise one or more factors known to be mitogenic for fibroblasts, such as one or more factors selected from the group consisting of: a) FGF-1; b) FGF-2; c) FGF-5; d) EGF; e) ciliary neurotrophic factor (CNTF); f) KGF-1; g) PDGF; h) platelet rich plasma; i) TGF-alpha; j) HGF-1; and k) a combination thereof.

[0169] Exosomes may or may not be obtained from fibroblasts while the fibroblasts are in a proliferating state. In some embodiments, exosomes are obtained from fibroblasts while the fibroblasts are cultured in a media comprising no proliferative factors or largely reduced levels of proliferation inducing growth factors and the growth factors may be undefined growth factors such as fetal calf serum, neonatal serum, cord blood serum, or platelet lysate, or the growth factors may be defined mitogens such as EGF, FGF-1, FGF-2, FGF-5. In someembodiments, exosomes may be obtained from fibroblasts cultured under hypoxia. In specific embodiments, exosomes are collected from fibroblasts that have been cultured in 2-8% oxygen for at least 1 day. The amount of oxygen may be 2, 3, 4, 5, 6, 7, or 8% in the culture. A range of oxygen levels in culture may be 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8%. In specific embodiments, the cells are cultured for 1- 15 days or 5-10 days. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days. The cells may be cultured for a range of days that is 1- 15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-14, 2-13, 2-12, 2- 11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-15, 5-14, 5-13, 5-12, 5- 11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-15, 10-14, 10-13, 10-12, 10-11, 11-15, 11-14, 11-13, 11-12, 12-15, 12-14, 12-13, 13- 15, 13-14, or 14-15 days. In culture, the cells may be passaged for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passages. In specific cases the fibroblasts are cultured in media selected from the group consisting of: a) Roswell Park Memorial Institute (RPMI-1640); b) Dublecco's Modified Essential Media (DMEM), c) Eagle's Modified Essential Media (EMEM), d) Optimem, and e) Iscove's Media.

[0170] In cases wherein exosomes from fibroblasts are utilized, the exosomes may be in a preparation, such as a preparation that comprises less than 5% polyethylene glycol. The exosomes may be purified using polyethylene glycol and / or using ultrafiltration. In some cases, polyethylene glycol is added to the exosomes after purification. The exosomes may or may not express one or more certain markers, such as markers selected from the group consisting of: a) CD63; b) CD9; c) major histocompatibility complex I (MHC I); d) CD56; and e) a combination thereof.

[0171] In some embodiments, fibroblasts are cultured using means known in the art for preserving viability and proliferative ability of fibroblasts. The disclosed methods may be applied both for individualized autologous exosome preparations and for exosome preparations obtained from established cell lines, for experimental or biological use. In some embodiments, chromatography separation methods are used for preparing membrane vesicles, particularly to separate the membrane vesicles from potential biological contaminants, wherein the microvesicles are exosomes, and cells utilized for generating said exosomes are fibroblast cells. The exosomes are obtained and may be prepared for administration to one or more individuals in need thereof.

[0172] In some embodiments membrane vesicles, particularly exosomes, may be purified, and possess an ability to promote aphthous ulcer healing. In one embodiment, a strong or weak, preferably strong, anion exchange may be performed. In addition, in a specific embodiment, the chromatography is performed under pressure. Thus, more specifically, it may consist of high performance liquid chromatography (HPLC). Different types of supports may be used to perform the anion exchange chromatography. More preferably, these may include cellulose, poly(styrene-divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycolmethacrylate co-polymer, or mixtures thereof, e.g., agarose-dextran mixtures. To illustrate this, it is possible to mention the different chromatography equipment composed of supports as mentioned above, particularly the following gels: SOURCE POROS® SEPHAROSE®, SEPHADEX®, TRISACRYL®, TSK-GEL SW OR PW®, SUPERDEX® TO YOPEARL HW and SEPHACRYL®, for example, which are suitable for the application of this invention. Therefore, in a specific embodiment, this invention relates to a method of preparing membrane vesicles, particularly exosomes, from a biological sample such as a tissue culture containing fibroblasts, comprising at least one step during which the biological sample is treated by anion exchange chromatography on a support selected from cellulose, poly(styrene-divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer, alone or in mixtures, optionally functionalized.

[0173] In addition, to improve the chromatographic resolution, within the scope of the disclosure, one can use supports in bead form. In particular embodiments, these beads have a homogeneous and calibrated diameter, with a sufficiently high porosity to enable the penetration of the objects under chromatography (i.e., the exosomes). In this way, given the diameter of exosomes (generally between 50 and 100 nm), to practice the invention, one can use high porosity gels, particularly between 10 nm and 5 pm, such as between approximately 20 nm and approximately 2 pm, including between about 100 nm and about 1 pm. For the anion exchange chromatography, the support used may be functionalized using a group capable of interacting with an anionic molecule. Generally, this group comprises an amine that may be ternary or quaternary, which defines a weak or strong anion exchanger, respectively. Within the scope of this disclosure, one can utilize a strong anion exchanger. In this way, according to the disclosure, a chromatography support as described above, functionalized with quaternary amines, may be used. Therefore, according to a more specific embodiment of the disclosure, the anion exchange chromatography is performed on a support functionalized with a quaternary amine. In specific cases, this support is selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran and silica, alone or in mixtures, and may be functionalized witha quaternary amine. Examples of supports functionalized with a quaternary amine include the gels SOURCEQ. MONO Q, Q SEPHAROSE®, POROS®HQ and POROS® QE, FRACTOGEL®TMAE type gels and TOYOPEARL SUPER®Q gels.

[0174] One example of a support to perform the anion exchange chromatography comprises poly(styrene-divinylbenzene). An example of this type of gel that may be used within the scope of this disclosure is SOURCE Q gel, particularly SOURCE 15 Q (Pharmacia). This support offers the advantage of very large internal pores, thus offering low resistance to the circulation of liquid through the gel, while enabling rapid diffusion of the exosomes to the functional groups, which are particularly important parameters for exosomes given their size. The biological compounds retained on the column may be eluted in different ways, particularly using the passage of a saline solution gradient of increasing concentration, e.g. from 0 to 2 M. A sodium chloride solution may particularly be used, in concentrations varying from 0 to 2 M, for example. The different fractions purified in this way may be detected by measuring their optical density (OD) at the column outlet using a continuous spectro-photometric reading. As an indication, under the conditions used in the examples, the fractions comprising the membrane vesicles were eluted at an ionic strength comprised between approximately 350 and 700 mM, depending on the type of vesicles.

[0175] Different types of columns may be used to perform this chromatographic step, according to requirements and the volumes to be treated. For example, depending on the preparations, it is possible to use a column from approximately 100 pL up to 10 mL or greater. In this way, the supports available have a capacity which may reach 25 mg of proteins / mL, for example. For this reason, a 100 pL column has a capacity of approximately 2.5 mg of proteins which, given the samples in question, allows the treatment of culture supernatants of approximately 2 L (which, after concentration by a factor of 10 to 20, for example, represent volumes of 100 to 200 mL per preparation). It is understood that higher volumes may also be treated, by increasing the volume of the column, for example. In addition, to practice this invention, it is also possible to combine the anion exchange chromatography step with a gel permeation chromatography step. In this way, according to a specific embodiment of the disclosure, a gel permeation chromatography step is added to the anion exchange step, either before or after the anion exchange chromatography step. In this embodiment, the permeation chromatography step takes place after the anion exchange step. In addition, in a specific variant, the anion exchange chromatography step is replaced by the gel permeation chromatography step. The present application demonstrates that membrane vesicles may also be purified using gel permeation liquid chromatography, particularly when this step is combined with an anionexchange chromatography or other treatment steps of the biological sample, as described in detail below.

[0176] To perform the gel permeation chromatography step, a support selected from silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer or mixtures thereof, e.g., agarose-dextran mixtures, may be used. As an illustration, for gel permeation chromatography, a support such as SUPERDEX®200HR (Pharmacia), TSK G6000 (TosoHaas) or SEPHACRYL®S (Pharmacia) may be used. The process according to the disclosure may be applied to different biological samples. In particular, these may comprise a biological fluid from a subject (bone marrow, peripheral blood, etc.), a culture supernatant, a cell lysate, a pre-purified solution or any other composition comprising membrane vesicles.

[0177] In this respect, in a specific embodiment of the disclosure, the biological sample is a culture supernatant of membrane vesicle-producing fibroblast cells.

[0178] In addition, according to one embodiment of the disclosure, the biological sample is treated, prior to the chromatography step, to be enriched with membrane vesicles (enrichment stage). In this way, in a specific embodiment, this disclosure relates to a method of preparing membrane vesicles from a biological sample, characterized in that it comprises at least: a) an enrichment step, to prepare a sample enriched with membrane vesicles, and b) a step during which the sample is treated by anion exchange chromatography and / or gel permeation chromatography.

[0179] In one embodiment, the biological sample is a culture supernatant treated so as to be enriched with membrane vesicles. In particular, the biological sample may be comprised of a pre-purified solution obtained from a culture supernatant of a population of membrane vesicle-producing cells or from a biological fluid, by treatments such as centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography, particularly with clarification and / or ultrafiltration and / or affinity chromatography. Therefore, one method of preparing membrane vesicles according to this disclosure more particularly comprises the following steps: a) culturing a population of membrane vesicle (e.g., exosome) producing cells under conditions enabling the release of vesicles, b) a step of enrichment of the sample in membrane vesicles, and c) an anion exchange chromatography and / or gel permeation chromatography treatment of the sample.

[0180] As indicated above, the sample (e.g., supernatant) enrichment step may comprise one or more centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography steps on the supernatant. In some embodiments, the enrichment step comprises (i) the elimination of cells and / or cell debris (clarification), optionally followed by(ii) a concentration and / or affinity chromatography step. In one specific embodiment, the enrichment step comprises an affinity chromatography step, optionally preceded by a step of elimination of cells and / or cell debris (clarification). An example of an enrichment step according to this disclosure comprises (i) the elimination of cells and / or cell debris (clarification), (ii) a concentration and (iii) an affinity chromatography. The cells and / or cell debris may be eliminated by centrifugation of the sample, for example, at a low speed, preferably below 1000 g, between 100 and 700 g, for example. Preferred centrifugation conditions during this step are approximately 300 g or 600 g for a period between 1 and 15 minutes, for example.

[0181] The cells and / or cell debris may also be eliminated by filtration of the sample, possibly combined with the centrifugation described above. The filtration may particularly be performed with successive filtrations using filters with a decreasing porosity. For this purpose, filters with a porosity above 0.2 pm, e.g., between 0.2 and 10 pm, may be used. It is particularly possible to use a succession of filters with a porosity of 10 pm, 1 pm, 0.5 pm followed by 0.22 pm.

[0182] A concentration step may also be performed, such as in order to reduce the volumes of sample to be treated during the chromatography stages. In this way, the concentration may be obtained by centrifugation of the sample at high speeds, e.g., between 10,000 and 100,000 g, to cause the sedimentation of the membrane vesicles. This may comprise a series of differential centrifugations, with the last centrifugation performed at approximately 70,000 g. The membrane vesicles in the pellet obtained may be taken up with a smaller volume and in a suitable buffer for the subsequent steps of the process. The concentration step may also be performed by ultrafiltration. In fact, this ultrafiltration allows both to concentrate the supernatant and perform an initial purification of the vesicles. According to a particular embodiment, the biological sample (e.g., the supernatant) is subjected to an ultrafiltration, preferably a tangential ultrafiltration. Tangential ultrafiltration comprises concentrating and fractionating a solution between two compartments (filtrate and retentate), separated by membranes of determined cut-off thresholds. The separation is carried out by applying a flow in the retentate compartment and a transmembrane pressure between this compartment and the filtrate compartment. Different systems may be used to perform the ultrafiltration, such as spiral membranes (Millipore, Amicon), flat membranes or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF, Sepracor). Within the scope of the invention, the use of membranes with a cut-off threshold below 1000 kDa, preferably between 300 kDa and 1000 kDa, or even more preferably between 300 kDa and 500 kDa, is advantageous.

[0183] The affinity chromatography step can be performed in various ways, using different chromatographic support and material. It is advantageously a non-specific affinity chromatography, aimed at retaining (i.e., binding) certain contaminants present within the solution, without retaining the objects of interest (i.e., the exosomes). It is therefore a negative selection. In some cases, an affinity chromatography on a dye may be used, allowing the elimination (i.e., the retention) of contaminants such as proteins and enzymes, for instance albumin, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or also co-factors, etc. More preferably, the support used for this chromatography step is a support as used for the ion exchange chromatography, functionalized with a dye. As a specific example, the dye may be selected from Blue SEPHAROSE®(Pharmacia), YELLOW 86, GREEN 5 and BROWN 10 (Sigma). The support may be agarose. It should be understood that any other support and / or dye or reactive group allowing the retention (binding) of contaminants from the treated biological sample can be used in the instant disclosure.

[0184] In one embodiment a membrane vesicle preparation process within the scope of this disclosure comprises the following steps: a) the culture of a population of membrane vesicle (e.g., exosome) producing cells under conditions enabling the release of vesicles, b) the treatment of the culture supernatant with at least one ultrafiltration or affinity chromatography step, to produce a biological sample enriched with membrane vesicles (e.g., with exosomes), and c) an anion exchange chromatography and / or gel permeation chromatography treatment of the biological sample. In a certain embodiment, step b) above comprises a filtration of the culture supernatant, followed by an ultrafiltration, such as tangential. In another embodiment, step b) above comprises a clarification of the culture supernatant, followed by an affinity chromatography on dye, such as on Blue SEPHAROSE®.

[0185] In addition, after step c), the material harvested may, if applicable, be subjected to one or more additional treatment and / or filtration stages d), particularly for sterilization purposes. For this filtration treatment stage, filters with a diameter less than or equal to 0.3 pm may be used, or for example, less than or equal to 0.25 pm. Such filters have a diameter of 0.22 pm, for example.

[0186] After step d), the material obtained is, for example, distributed into suitable devices such as bottles, tubes, bags, syringes, etc., in a suitable storage medium. The purified vesicles obtained in this way may be stored cold, frozen or used extemporaneously. Therefore, a specific preparation process within the scope of the disclosure comprises at least the following steps: c) an anion exchange chromatography and / or gel permeation chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, of the materialharvested after stage c). In a first variant, the process according to the disclosure comprises: c) an anion exchange chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). In some cases, instead of being stored the material may be used for one or more individuals in the absence of a prior storage step.

[0187] In another variant, the process according to the disclosure comprises: c) a gel permeation chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). According to a third variant, the process according to the disclosure comprises: c) an anionic exchange treatment of the biological sample followed or preceded by gel permeation chromatography, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c).V. Delivery of Therapeutic Factors

[0188] In some embodiments, the fibroblasts, modified fibroblasts, fibroblast derived factors which can be extracted and / or concentrated, are formulated into a pharmaceutical composition capable of inducing benefit. In some embodiments, the therapeutic factors are incorporated into lozenges, mouthwash, lollipop-like structures, chewing gum, toothpaste, or oral dissolving tablets for local administration to the affected oral mucosa. Other delivery modalities, such as gels, sprays, or patches, may also be employed depending on the clinical context and patient preference (16).

[0189] In some embodiments, compositions of the disclosure may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the therapeutic factors comprise factors derived from fibroblasts, fibroblast organoids, from culture of fibroblasts with other cells, or from culture of fibroblast organoids with other cells. In some embodiments, therapeutic factors comprise extracellular matrix factors, vasoconstriction regulating factors, antimicrobial factors, extracellular remodeling factors, growth factors, cytokines, inflammation modulating factors, chemokines, and / or clotting factors, for example, but not limited to collagens, glycosaminoglycans, hyaluronic acid, elastin, laminins, tenascins, fibronectin, endothelin, catecholamines, epinephrine, norepinephrine, prostaglandins, bradykinin, fibrinopeptide, serotonin, histamine, thromboxane A2, myeloperoxidase, azurocidin, lysozyme, bacterial permeability increasing protein, cathepsin G, elastase, protease 3, humancationic antimicrobial protein 18, lactoferrin, matrix metalloprotease 8, collagenase 2, chymase, tryptase, matrix metalloproteases, tissue inhibitor of metalloproteinases- 1, -2, -3, angiopoietins, FGF, KGF, VEGF, PDGF, TGF, EGF, IGF, TNF-alpha, IL-6, IL 1 -beta, INF- alpha, INF -beta, IL-2, IL-4, IL-8, IL- 10, IL-7, hyaluronan, osteopontin, periostin, vitronectin, angiotensin, CCN2, Cx43, CXCL8, CXCL1, CXCL12, CXCL2, RANTES, MCP, MIP, lymphotactin, CCL2, CXCL10, fibrinogen, von Willebrand factor, collagen, fibrin, biglycan, decorin, versican, heparan sulfate, tenascin C, uric acid, SI 00 protein, ATP, F-actin, cyclophilin A, histones, HMGB1, IL-1 alpha, IL-33, SAP130, DNA, RNA, mtDNA, formyl peptide, mROS, calreticulin, defensins, cathelici din, eosinophil-derived neurotoxin, granulysin, syndecans, glypicans, mast cell protease-4 or -5, CCN1, stromal derived factor-1 (SDF-1 / CXCL12), monocyte chemoattractant protein-1 (MCP-1), Tie-2, SKINTs, CD100, ICAM-1, VCAM-1, P-selectin, E-selectin.A) Carriers

[0190] In some embodiments, pharmaceutical compositions of the present disclosure comprise an effective amount of one or more compositions comprising fibroblasts, modified fibroblasts, and / or products derived from fibroblasts, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical” and “pharmacologically acceptable” and used interchangeably herein refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, such as, for example, a human, as appropriate, and do not interfere with the therapeutic methods of the disclosure. The preparation of a pharmaceutical composition that comprises fibroblasts, modified fibroblasts, and / or products derived from fibroblasts, or additional active ingredient(s), will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, specifically incorporated by reference herein in its entirety. Moreover, for administration to a subject, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0191] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents,flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, specifically incorporated by reference herein in its entirety). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. The compositions comprising fibroblasts, modified fibroblasts, activated fibroblasts, products derived therefrom, or a combination thereof may comprise different types of carriers depending on whether it is to be administered in solid or liquid form.

[0192] Further in accordance with the present disclosure, the composition of the present disclosure suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and include liquid, semisolid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in practicing the methods of the present disclosure is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, alcohols, and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0193] In accordance with the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art. The compositions comprising fibroblasts, modified fibroblasts, activated fibroblasts, products derived therefrom, or a combination thereof may be lyophilized.

[0194] In a specific embodiment of the present disclosure, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of biological activity. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0195] In further embodiments, the present disclosure may include the use of a pharmaceutical lipid vehicle compositions that incorporate compositions comprising fibroblasts, modified fibroblasts, activated fibroblasts, products derived therefrom, or a combination thereof, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present disclosure.

[0196] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the composition(s) comprising fibroblasts, modified fibroblasts, and / or products derived from fibroblasts may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0197] The composition(s) comprising fibroblasts, modified fibroblasts, and / or products derived from fibroblasts, may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

[0198] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0199] For optimal oral administration, the composition(s) comprising fibroblasts, modified fibroblasts, and / or products derived from fibroblasts, of the present disclosure may be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, lollipop-like structure, chewing gum, oral spray, lozenge, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell’s Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.Lozenge

[0200] As described herein, a “lozenge” includes at least a factor of this disclosure (e.g., fibroblasts, modified fibroblasts, activated fibroblasts, products derived therefrom, (e.g., exosomes, lysates, apoptotic bodies), or a combination thereof) and a soluble-fiber matrix. Therapeutic factors can be dispersed in the soluble-fiber matrix such that the therapeutic factors are released from the lozenge as it dissolved when the lozenge is received within the oral cavity and exposed to saliva.

[0201] In some embodiments, the lozenge may include one or more additives adapted to be released from the lozenge when it is placed in the oral cavity. In some embodiments, the additives may comprise sweeteners (e.g., maltitol, mannitol, xylitol, sucralose, etc.), solvents (e.g., water), flavorants, binders, colorants, fillers, plasticizers, antioxidants, preservatives, buffers, pH adjusting agents, and / or processing aids. Fillers may be included in the solublefiber matrix to alter the texture or pliability of the lozenge. The soluble-fiber matrix can also include plasticizers (e.g., propylene glycol, glycerol), which can increase the softness of a lozenge provided herein. Antioxidants may be used to preserve the therapeutic factors in a lozenge provided herein. Processing aids may be included to facilitate the shaping process of a lozenge provided herein.

[0202] In some embodiments, a lozenge provided herein can take up to 1 hour to dissolve when placed in an individual’s mouth. In some embodiments, a lozenge provided herein can take between 1 minute and 30 minutes to dissolve in an individual’s mouth. In some embodiments, a lozenge provided herein can take between 2 minutes and 15 minutes to dissolve in an individual’s mouth.VI. Pharmaceutical Compositions

[0203] In certain aspects, the compositions or agents for use in the methods, such as therapeutic fibroblasts, CAR-expressing fibroblasts, etc., are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e., to a specific location of the body, such as an ulcer) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration.

[0204] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer’s solutions, dextrose solution, Hank’s solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0205] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0206] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0207] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0208] In certain aspects, the pharmaceutical compositions are administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0209] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water,alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer’s dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0210] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral delivery. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0211] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0212] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

[0213] In some embodiments, a single administration of cells is provided. In some embodiments, multiple administrations are provided. In some embodiments, multiple administrations are provided over the course of 3-7 consecutive days. In some embodiments, 3-7 administrations are provided over the course of 3-7 consecutive days. In some embodiments, 5 administrations are provided over the course of 5 consecutive days. In some embodiments, a single administration of between about 105and about 1013cells per 100 kg is provided. In some embodiments, a single administration of between about 1.5xl08and about 1.5xl012cells per 100 kg is provided. In some embodiments, a single administration of between about IxlO9and about 5xl0ncells per 100 kg is provided. In some embodiments, a single administration of about 5xl010cells per 100 kg is provided. In some embodiments, a singleadministration of IxlO10cells per 100 kg is provided. In some embodiments, multiple administrations of between about 105and about 1013cells per 100 kg are provided. In some embodiments, multiple administrations of between about 1.5xl08and about 1.5xl012cells per 100 kg are provided. In some embodiments, multiple administrations of between about IxlO9and about 5xl0ncells per 100 kg are provided over the course of 3-7 consecutive days. In some embodiments, multiple administrations of about 4xl09cells per 100 kg are provided over the course of 3-7 consecutive days. In some embodiments, multiple administrations of about 2xlOncells per 100 kg are provided over the course of 3-7 consecutive days. In some embodiments, 5 administrations of about 3.5xl09cells are provided over the course of 5 consecutive days. In some embodiments, 5 administrations of about 4xl09cells are provided over the course of 5 consecutive days. In some embodiments, 5 administrations of about 1.3xl0ncells are provided over the course of 5 consecutive days. In some embodiments, 5 administrations of about 2xlOncells are provided over the course of 5 consecutive days.VII. Cancer Therapy

[0214] In some aspects, the disclosed methods comprise treating aphthous ulcers of an individual undergoing cancer therapy. In some aspects, the cancer therapy comprises a local or systemic cancer therapy. In some aspects, the cancer therapy comprises both a local and systemic cancer therapy. In some aspects, the cancer therapy comprises a radiotherapy, chemotherapy, immunotherapy, or other cancer therapy. The individual may be receiving a combinations of these therapies.A) Radiotherapy

[0215] In some aspects, an individual is receiving radiotherapy, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A non-limiting example of ionizing radiation is x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0216] In some aspects, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some aspects, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some aspects, the internalradiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some aspects, the radiotherapy is administered to a primary tumor.

[0217] In some aspects, the amount of ionizing radiation is greater than 20 gray (Gy) and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein). In some aspects, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the dose may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0218] In some aspects, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of radiation is at least, at most, or about 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some aspects, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some aspects, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.B) Chemotherapies

[0219] In some aspects, an individual is being administered, has been administered, or is planned to be administered a chemotherapy. Classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5- fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g, vinblastine, vincristine), epipodophylotoxins (e.g, etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhy diazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane).

[0220] In some aspects, an individual is being administered, has been administered, or is planned to be administered cisplatin. Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.

[0221] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). Doxorubicin is absorbed poorly and is preferably administered intravenously.

[0222] Nitrogen mustards are another suitable chemotherapeutic agent. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria. Because of adverse gastrointestinal effects, the intravenous route is preferred in certain cases. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0223] Additional chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode-oxyuridine; FudR).C) Cancer Immunotherapy

[0224] In some aspects, an individual is being administered a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can, in some cases, be categorized as active, passive or hybrid (i.e., active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes, and / or cytokines. Various immunotherapies are known in the art, and certain examples are described below. i) Checkpoint Inhibitors and Combination Treatment

[0225] In some embodiments, an individual is being administered, has been administered, or is planned to be administered one or more immune checkpoint inhibitors. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy,” “checkpoint blockade therapy,” “immune checkpoint therapy,” “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer. a) PD-1, PDL1, and PDL2 inhibitors

[0226] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0227] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0228] In some aspects, an individual is being administered, has been administered, or is planned to be administered an immune checkpoint inhibitor that targets PD-1. The PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0229] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO201 1 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0230] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinationsthereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0231] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. b) CTLA-4, B7-1, and B7-2

[0232] In some aspects, an individual is being administered, has been administered, or is planned to be administered an immune checkpoint inhibitor that targets the cytotoxic T- lymphocyte-associated protein 4 (CTLA-4 or CTLA4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7- 2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7- 1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0233] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0234] Other anti-CTLA-4 antibodies recognized in the art are contemplated. For example, the anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0235] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0236] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in some aspects, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In some aspects, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. c) LAG3

[0237] In some aspects, an individual is being administered, has been administered, or is planned to be administered an immune checkpoint inhibitor that targets the lymphocyteactivation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has the Genbank accession number NM 002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+T cells in a tolerogenic state and, working with PD- 1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors being administered to an individual may block one or more functions of LAG3 activity.

[0238] In some aspects, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0239] Anti-LAG3 antibodies are well known in the art. For example, anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as LAG3.1); WO 2016 / 028672 (MK-4280, also known as 28G-10); WO 2017 / 019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11 :6 (LBL-007) are contemplated. These and other anti-LAG-3 antibodies can be found in, for example: WO 2016 / 028672, WO 2017 / 106129, WO 2017062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 220569, WO 2018 / 071500, WO 2017 / 015560; WO 2017 / 025498, WO 2017 / 087589 , WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271, WO 2017 / 086367, WO 2017 / 086419, WO 2018 / 034227, and WO 2014 / 140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also are contemplated.

[0240] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d) TIM-3

[0241] In some aspects, an individual is being administered, has been administered, or is planned to be administered an immune checkpoint inhibitor that targets the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782. TIM-3 is found on the surface fFNy-producing CD4+Thl and CD8+Tel cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates T-cell exhaustion. TIM-3 has also been shown as a CD4+Thl-specific cell surface protein that regulates macrophage activation. A checkpoint inhibitor may block one or more functions of TIM-3 activity.

[0242] In some aspects, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0243] Anti-TIM-3 antibodies are known in the art. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367. Other anti-TIM- 3 antibodies can be found in, for example: US 9,605,070, US 8,841,418, US2015 / 0218274, and US 2016 / 0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 are also contemplated.

[0244] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies. ii) Activator of co-stimulatory molecules

[0245] In some aspects, an individual is being administered, has been administered, or is planned to be administered an immunotherapy that comprises an activator (also “agonist”) of a co-stimulatory molecule. In some aspects, the agonist comprises an agonist of CD3, B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Agonists include activating antibodies, polypeptides, compounds, and nucleic acids.iii) Dendritic cell therapy

[0246] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0247] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0248] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0249] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0250] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0251] In some embodiments, an individual is being administered, has been administered, or is planned to be administered dendritic cell therapy. iv) Chimeric Immune Receptors (CIR)

[0252] Chimeric immune receptors (CIRs), including Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors), and chimeric T cell receptors (cTCRs; also known as artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of an antigen binding domain, e.g., an antibody, onto a T cell, natural killer (NK) cell, or other immune cell.The receptors are called chimeric because they are fused of parts from different sources. CIR- immune cell therapy, including CAR-T cell therapy and cTCR-T cell therapy, refers to a treatment that uses such transformed cells for cancer therapy, where the transformed cells are immune cells. Similar therapies include, for example, CAR-NK or cTCR-NK cell therapy, which uses engineered NK cells.

[0253] The basic principle of CIR cell design involves recombinant receptors that combine antigen-binding and immune cell activating functions, e.g., T-cell activating functions. The general premise of CIR cells is to artificially generate immune cells targeted to markers found on cancer cells. Medical professionals can remove immune cells, e.g., T or NK cells, from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the immune cell has been engineered to become a CIR-immune cell, it acts as a “living drug”. CIR-immune cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates immune cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CIR-immune cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CIR-immune cells is determined by the choice of molecule that is targeted.

[0254] Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).

[0255] While CARs are encouraging therapies against cancer, CARs are limited to surface antigens and have limited stimulatory capabilities. TCRs have properties that may help overcome some of the deficiencies of CARs. For example, TCRs can recognize any peptide that is processed for antigen presentation, and TCR signaling is 10 to 100 times more sensitive than CAR signaling (Harris et al., J Immunol. 2018 Feb l;200(3): 1088-1100.). For at least these reasons, TCR-based immunotherapy is a promising anti-cancer therapy, especially towards solid tumors.

[0256] TCRs are composed of aP (or y5) chain heterodimers that assemble at the cell membrane with the CD3 signaling complex (CD3sy, CD3s5, and CD3( ). The a and P chains each comprise an extracellular immunoglobulin (Ig)-like domain comprising a variable region that provides antigen binding specificity and a constant domain, a transmembrane domain, and a short cytoplasmic region that lacks intracellular signaling motifs. Upon TCR binding to an antigen, immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 complex undergo phosphorylation and activate a downstream T cell signaling cascade.

[0257] Chimeric TCRs (cTCRs) combine the specificity of antigen binding domains, e.g., antibody scFv, with the ability of TCRs to engage endogenous signaling complexes (e.g., CD3 complex) to activate immune cells, e.g., T cells. cTCRs may comprise various structural configurations. In general, the endogenous variable region of a and / or p chains are replaced with an antigen binding domain of interest (e.g., a scFv targeting a cancer antigen) that is linked directly to the constant region of the a or P chains. In some instances, the entire antigen binding domain may be linked to one or both a and P chains, although the antigen binding domain may also be split amongst the two chains (e.g., an antibody VL chain may be linked to the a-chain while the VH may be linked to the P chain, or vice versa). In some instances, the a and / or p chains may be engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8): 3898- 3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety. The antigen binding domain may also be linked to other components of the TCR complex, such as the y5 chains or the CD3 complex subunits, including the CD3y, CD35, and / or CD3s subunits.

[0258] In some embodiments, an individual is being administered, has been administered, or is planned to be administered a CIR therapy. v) Cytokine therapy

[0259] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0260] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IF NX).

[0261] Interleukins have an array of immune system effects. IL-2 is an example of interleukin cytokine therapy.

[0262] In some embodiments, an individual is being administered, has been administered, or is planned to be administered a cytokine therapy. vi) Adoptive cell therapy

[0263] Adoptive cell therapy is a form of passive immunization by the transfusion of immune cells, such as T cells, NK cells, or other immune cells (also called “adoptive cell transfer”). Immune cells used for adoptive cell therapy include those found in normal tissue and those found in tumor tissue (where they are known as tumor infiltrating immune cells or tumor infiltrating lymphocytes). Although tumor infiltrating immune cells can attack a tumor, the environment within the tumor is generally highly immunosuppressive, preventing immune- mediated tumor death.

[0264] Multiple ways of producing and obtaining tumor targeted immune cells have been developed. Immune cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing may be performed ex vivo, with the results reinfused. Activation can take place through gene therapy, by exposing the immune cells to tumor antigens, or by other methods known in the art.

[0265] In some embodiments, an individual is being administered, has been administered, or is planned to be administered an adoptive cell therapy. vii) T-cell Engager (TCE)

[0266] T-cell engagers (TCEs) are chimeric antibodies comprising a target cell binding domain comprising an antigen binding domain such as a single-domain antibody (e.g., a VHH) that specifically binds to an antigen on a target cell, and an immune effector cell binding domain that specifically binds to an antigen on an immune effector cell. In this way, TCEs can redirect T cell to recognize and kill tumor cells.

[0267] In certain embodiments, an individual is being administered, has been administered, or is planned to be administered a T-cell engager.D) Oncolytic virus

[0268] In some aspects, the cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy. One example of oncolytic virus therapy is teserpaturev (Delytact®).

[0269] In some aspects, an individual is being administered, has been administered, or is planned to be administered an oncolytic virus.E) Hormone therapy

[0270] In some aspects, a cancer therapy of the present disclosure is a hormone therapy. In particular aspects, a prostate cancer therapy comprises hormone therapy. Various hormone therapies are known in the art and contemplated herein. Examples of hormone therapies include, but are not limited to, luteinizing hormone-releasing hormone (LHRH) analogs, LHRH antagonists, androgen receptor antagonists, and androgen synthesis inhibitors.

[0271] In some embodiments, an individual is being administered, has been administered, or is planned to be administered a hormone therapy.F) Surgery

[0272] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0273] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0274] In some embodiments, an individual is undergoing, has undergone, or is planned to undergo surgery.G) Additional cancer therapies

[0275] Therapeutic methods disclosed herein may comprise one or more additional cancer therapies. A cancer therapy of the disclosure may comprise, for example, cryoablative therapy, high-intensity ultrasound (also “high-intensity focused ultrasound”), photodynamic therapy, laser ablation, and / or irreversible electroporation. A cancer therapy of the disclosure may comprise 1, 2, 3, 4, 5, or more distinct therapeutic methods.

[0276] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, aspects of the present disclosure include individuals that have been previously treated with a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the individual is one that has been determined to be resistant to the therapy described herein. In some aspects, the individual is one that has been determined to be sensitive to the therapy described herein.

[0277] In some embodiments, an individual is being administered, has been administered, or is planned to be administered an additional cancer therapy.VIII. Kits of the Disclosure

[0278] Any of the cellular and / or non-cellular compositions described herein or similar thereto may be comprised in a kit. In a non-limiting example, one or more compositions for use in methods for treating aphthous ulcers may be comprised in a kit. Such compositions may include cells; factors derived from cells; carriers, and so forth. The kit may comprise additional components, such as medicaments for treating aphthous ulcers and / or cancer. In some embodiments, the medicaments for treating aphthous ulcers may comprise oral care solutions (e.g., toothpaste, saline solutions, and / or cleaning mouthwash), pain relievers (e.g., menthol, benzocaine, lidocaine, morphine), immunomodulators (e.g., dexamethasone, clobetasol, fluocinonide, pentoxifylline, dapsone, and / or thalidomide), and / or antiseptic agents (e.g., chlorhexidine gluconate, alcohol, benzalkonium chloride, and / or hydrogen peroxide). In some embodiments, the medicaments to treat cancer may comprise chemotherapy, immunotherapy, oncolytic virus therapy, and / or hormone therapy.

[0279] Some components of the kits may be packaged either in solid, semi-solid, or liquid form, e.g., in tablets, mouthwash, lollipop-like structures, chewing gum, spray, syrups, wafers, troches, pills, etc. The components are provided in suitable containers, e.g., bottles, wrappers, pouches, bags, blister packaging, or other container means into which a component may be placed, and preferably, suitably portioned to comprise a preferred dose. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single container. In some embodiments, the kit comprises a composition of the disclosure and an aphthous ulcer therapy. In some embodiments, the kit comprises a composition of the disclosure and a cancer therapy.

[0280] The kits of the present disclosure also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired containers are retained.

[0281] Some components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent.EXAMPLES

[0282] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Lozenges comprising fibroblasts or fibroblasts derived factors promote aphthous ulcer recovery in individuals undergoing radiotherapy

[0283] In one embodiment, a group patients with neck and head cancer are recruited for a clinical trial to test the efficacy of lozenges in promoting recovery of aphthous ulcers caused by radiotherapy. A first group are allocated to a control group that receives standard care (i.e., conventional mouthwash, pain management medication, anti-inflammatory medication) and non-medicated lozenges. A second group is allocated to treatment group 1 that receives standard care and lozenges comprising fibroblasts. A third group is allocated to treatment group 2 that receives standard care and lozenges comprising fibroblasts derived factors. The patients receive cancer care per their doctor approved treatment regime of radiation therapy. All individuals receive daily care to prevent or ameliorate aphthous ulcers.

[0284] All individuals are monitored through the entire procedure for evaluating the severity of their aphthous ulcers using the World Health Organization’s Oral Mucositis Scale (WHO-OMS) (0 (none) = none; 1 (mild) = oral soreness, erythema; II (moderate) = oral erythema, ulcers, solid diet is tolerated; III (severe) = oral ulcers, only liquid diet is possible; IV (life-threatening) = oral alimentation is impossible).Example 2 - Lozenges comprising fibroblasts or fibroblasts derived factors promote aphthous ulcer recovery in individuals undergoing chemotherapy

[0285] In one embodiment, a group of patients undergoing chemotherapy for solid tumors are recruited to test the efficacy of lozenges comprising fibroblasts or fibroblasts derived factors in the treatment of chemotherapy-induced aphthous ulcers. A first group of individuals are allocated to a control group that receives standard care (i.e., conventional mouthwash, pain management medication, anti-inflammatory medication) and non-medicated lozenges. A second group of individuals are allocated to treatment group 1 that receives standard care and lozenges comprising fibroblasts. The remaining individuals are allocated to treatment group 2 that receives standard care and lozenges comprising fibroblasts derived factors. All individuals are instructed to follow standard care procedures and take lozenges ad libitum. The patients receive cancer care per their doctor approved treatment regime of chemotherapy. All individuals are contacted daily to report the incidence and severity of aphthous ulcers and a medical professional assesses the WHO-OMS score during each chemotherapy visit.

[0286] Individuals are evaluated on levels of discomfort, incidence, pain, and complications related to aphthous ulcers. All individuals are monitored through the entire procedure for evaluating the severity of their aphthous ulcers using the World Health Organization’s Oral Mucositis Scale (WHO-OMS) (0 (none) = none; 1 (mild) = oral soreness, erythema; II (moderate) = oral erythema, ulcers, solid diet is tolerated; III (severe) = oral ulcers, only liquid diet is possible; IV (life-threatening) = oral alimentation is impossible).* * *

[0287] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCES

[0288] The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Sroussi, H. Y., et al., Common oral complications of head and neck cancer radiation therapy: mucositis, infections, saliva change, fibrosis, sensory dysfunctions, dental caries, periodontal disease, and osteoradionecrosis. Cancer Med, 2017. 6(12): p. 2918-2931.2. Cialdai, F., C. Risaliti, and M. Monici, Role of fibroblasts in wound healing and tissue remodeling on Earth and in space. Front Bioeng Biotechnol, 2022. 10: p. 958381.3. Petrof, G., et al., Fibroblast cell therapy enhances initial healing in recessive dystrophic epidermolysis bullosa wounds: results of a randomized, vehicle-controlled trial. Br J Dermatol, 2013. 169(5): p. 1025-33.4. Chuong, C.M., et al., What is the 'true' function of skin? Exp Dermatol, 2002. 11(2): p. 159-87.5. Bainbridge, P., Wound healing and the role of fibroblasts. J Wound Care, 2013. 22(8): p. 407-8, 410-12.6. Li, Z. and P. Maitz, Cell therapy for severe burn wound healing. Bums Trauma, 2018. 6: p. 13.7. Kubo, K. and Y. Kuroyanagi, A study of cytokines released from fibroblasts in cultured dermal substitute. Artif Organs, 2005. 29(10): p. 845-9.8. Spiekstra, S.W., et al., Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Regen, 2007. 15(5): p. 708-17.9. Guerrero-Juarez, C.F., et al., Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds. Nat Commun, 2019. 10(1): p. 650.10. Driskell, R.R., et al., Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature, 2013. 504(7479): p. 277-281.11. Nuschke, A., Activity of mesenchymal stem cells in therapies for chronic skin wound healing. Organogenesis, 2014. 10(1): p. 29-37.12. Guo, S. and L.A. Dipietro, Factors affecting wound healing. J Dent Res, 2010. 89(3): p. 219-29.13. Martin, P. and R. Nunan, Cellular and molecular mechanisms of repair in acute and chronic wound healing. Br J Dermatol, 2015. 173(2): p. 370-8.14. Liang, C.C., A.Y. Park, and J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc, 2007. 2(2): p. 329-33.15. Mooren, O.L., B.J. Galletta, and J. A. Cooper, Roles for actin assembly in endocytosis. Annu Rev Biochem, 2012. 81 : p. 661-86.16. Jorgensen, M.G., et al., Adipose-derived regenerative cells and lipotransfer in alleviating breast cancer-related lymphedema: An open-label phase I trial with 4 years of follow-up. Stem Cells Transl Med, 2021. 10(6): p. 844-854.17. Brown TJ, Gupta A. Management of Cancer Therapy-Associated Oral Mucositis. JCO Oncol Pract. 2020 Mar;16(3): 103-109.18. Sonis S, Treister N, Chawla S, Demetri G, Haluska F. Preliminary characterization of oral lesions associated with inhibitors of mammalian target of rapamycin in cancer patients. Cancer. 2010 Jan 1;116(l):210-5.19. Lalla RV, Bowen J, Barasch A, Elting L, Epstein J, Keefe DM, McGuire DB, Migliorati C, Nicolatou-Galitis O, Peterson DE, Raber-Durlacher JE, Sonis ST, Elad S; Mucositis Guidelines Leadership Group of the Multinational Association of Supportive Care in Cancer and International Society of Oral Oncology (MASCC / ISOO). MASCC / ISOO clinical practice guidelines for the management of mucositis secondary to cancer therapy. Cancer. 2014 May 15; 120(10): 1453-61.

Claims

CLAIMS:What is claimed is:

1. A method of treating one or more aphthous ulcers in an individual, the method comprising administering fibroblasts, fibroblasts organoids, and / or fibroblast-derived factors to the individual.

2. The method of claim 1, wherein the fibroblasts are dehydrated or lyophilized.

3. The method of claim 1 or 2, wherein the fibroblast-derived factors comprise exosomes, microvesicles, conditioned media, lysate, and / or apoptotic bodies.

4. The method of any one of claims 1-3, wherein the fibroblasts or fibroblast-derived factors are filtered, enriched, lysed, extracted, purified, and / or concentrated.

5. The method of any one of claims 1-4, wherein the fibroblast derived-factors comprise extracellular matrix proteins, growth factors, cytokines, and / or inhibitors of metalloproteinases.

6. The method of claim 5, wherein the extracellular matrix proteins comprise laminins, fibronectins, collagens, and / or elastic fibers.

7. The method of claim 5 or 6, wherein the growth factors or cytokines comprise bFGF, VEGF, HGF, PDGF, TGF-B1, KGF, IL-6, IL-8, or a combination thereof.

8. The method of any one of claims 1-7, wherein the fibroblasts are pre-activated.

9. The method of claim 8 wherein pre-activation of fibroblasts comprises culturing in hypoxic conditions, co-culturing with one or more cell types, culturing with one or more activating agents, or a combination thereof.

10. The method of claim 9 wherein the hypoxic conditions comprise 0.1%- 10%, 0. l%-5%, 0. l%-2.5%, or 0.1%-1% oxygen.

11. The method of claim 9 or 10, wherein the cell types for co-culture comprise platelets, neutrophils, monocytes, macrophages, Ml macrophages, M2 macrophages, M2a macrophages, dendritic cells, Langerhans cells, T cells, aP T cells, y5 T cells, T helper cells, regulatory T cells, killer T cells, endothelial cells, pericytes, hematopoietic progenitor cells, epidermal cells,epidermal stem cells, smooth muscle cells, lymphocytes, mast cells, NK cells, adipose stromal cells, immune cells, epithelial cells, stem cells, keratinocytes, or a combination thereof.

12. The method of any one of claims 9-11, wherein the pre-activating culture comprises chemical agents, RNA, micro-RNA, RNAi, DNA, viral nucleic acid, exosomes, cytokines, growth factors, hormones, platelet-rich plasma, platelet lysate, serum, serum replacement, or a combination thereof.

13. The method of claim 12, wherein the cytokine is selected from the group consisting of IFN-gamma, TNF-alpha, interleukin (IL)-l, IL-6, IL-7, IL-8, IL-12, IL-15, IL-17, IL-33, and a combination thereof14. The method of claim 12 or 13, wherein the growth factor is selected from the group consisting of FGF-1, VEGF, HGF, FGF, EGF, KGF, and a combination thereof15. The method of any one of claims 12-14, wherein the serum comprises umbilical cord blood serum, autologous serum, human serum, or a combination thereof.

16. The method of any one of claims 1-15, wherein the fibroblasts are autologous, allogeneic or xenogeneic with respect to the individual.

17. The method of any one of claims 1-16, wherein the fibroblasts are mammalian.

18. The method of any one of claims 1-17, wherein the fibroblasts are from human origin.

19. The method of any one of claims 1-18, wherein the fibroblasts are generated from stem cells, induced pluripotent stem cells, pluripotent stem cells, embryonic stem cells and / or non- embryonic stem cells.

20. The method of any one of claims 1-19, wherein the fibroblasts and / or fibroblast-derived materials are derived from skin, adipose tissue, bone marrow, umbilical cord, Wharton’s jelly, omentum, peripheral blood, mobilized peripheral blood, perivascular tissue; adipose tissue; placental tissue; amniotic membrane; omentum; dental pulp; fallopian tube tissue; hepatic tissue; renal tissue; cardiac tissue; tonsillar tissue; testicular tissue; ovarian tissue; neuronal tissue; auricular tissue; colonic tissue; submucosal tissue; hair follicle tissue; pancreatic tissue; skeletal muscle tissue; subepithelial umbilical cord tissue; foreskin tissue; or a combination thereof.

21. The method of any one of claims 1-20, wherein the fibroblasts comprise expression of c-Kit, hes related family bHLH transcription factor with YRPW motif 1 (Heyl), a-smooth muscle actin (SMA), Vimentin (including intracellular or extracellular vimentin), Cyclin D2, Snail, E-cadherin, NK2 homeobox 5 (Nkx2.5), GATA binding protein 4 (GATA4), cluster of differentiation (CD) 105, CD271, CD90, CD29, CD73, CD44, CD10, CD13, CD44, Wilms’ tumor 1 (Wtl), CXC motif chemokine receptor 4 (CXCR-4), fibroblast growth factor 1 (FGF- 1) receptor, stage specific embryonic antigen 3 (SSEA-3), tumor necrosis factor alpha (TNF- a) receptor- 1, toll like receptor 4 (TLR4), receptor for acetylated end products (RAGE), hepatocyte growth factor (HGF), express octamer-binding transcription factor 4 (Oct-4), CD- 34, Kriippel-like factor 4 (KLF-4), Nanog, Sox-2, Rex-1, growth differentiation factor 3 (GDF- 3), Stella, possess enhanced expression of GDF-11, or a combination thereof.

22. The method of any one of claims 1-21, wherein the fibroblast and / or fibroblast-derived materials are comprised in a pharmaceutically acceptable excipient.

23. The method of any one of claims 1-22, wherein the fibroblast and / or fibroblast-derived materials are comprised in a lozenge, mouthwash, film, lollipop-like structure, chewing gum, or dissolving tablet.

24. The method of claim 23, wherein the lozenge, mouthwash, lollipop-like structure, chewing gum, or dissolving tablet further comprise sweeteners, solvents, flavorants, binders, colorants, fillers, plasticizers, antioxidants, preservatives, buffers, pH adjusting agents, processing aids, adjuvants, or a combination thereof.

25. The method of any one of claims 1-24, further comprising administering fibroblasts and / or fibroblast-derived factors to the mouth of the individual.

26. The method of any one of claims 1-25, wherein the individual suffers from infection- induced, gastrointestinal disorder-induced, mechanical injury-induced, hormone imbalance- induced, sensitivity-induced, medicament-induced, and / or cancer therapy-induced aphthous ulcers.

27. The method of any one of claims 1-26, wherein the individual is undergoing cancer therapy.

28. The method of claims 26 or 27, wherein the cancer therapy comprises chemotherapy, radiation therapy, immunotherapy, oncolytic virus therapy, hormone therapy, surgery, or a combination thereof.

29. The method of any one of claims 1-28, wherein administration of the fibroblasts and / or fibroblast-derived factors occurs before, during, and / or after a cancer therapy.

30. The method of any one of claims 1-29, wherein administration of the fibroblasts and / or fibroblast-derived factors occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in a 24 hour period.

31. The method of any one of claims 1-30, wherein administration of the fibroblasts and / or fibroblast-derived factors occurs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more continuous or discontinuous days.

32. The method of any one of claims 1-31, wherein the individual has recurrent aphthous ulcers.

33. The method of any one of claims 1-32, wherein the aphthous ulcers are minor or major.

34. The method of any one of claims 1-33, wherein the aphthous ulcers have persisted for more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more weeks during or after a cancer therapy but before treatment with the fibroblasts and / or fibroblast-derived factors.

35. The method of any one of claims 1-34, wherein the fibroblasts and / or fibroblast- derived factors are delivered in a kit.

36. A kit comprising fibroblast and / or fibroblasts derived factors in a suitable form for buccal delivery and one or more additional treatment(s).

37. The kit of claim 36, wherein the suitable form for buccal delivery comprises a lozenge, mouthwash, lollipop-like structure, chewing gum, or dissolving tablet.

38. The kit of claim 36 or 37, wherein the additional treatment comprises an oral care solution, pain reliever, immunomodulator medicament, anti-septic agent, anti-bacterial agent, anti-fungal agent, cancer therapy, or a combination thereof.

39. The kit of claim 38, wherein the oral care solutions comprise toothpaste, saline solution, and / or cleaning mouthwash.

40. The kit of claim 38 or 39, wherein the immunomodulator medicament comprises dexamethasone, clobetasol, fluocinonide, pentoxifylline, dapsone, and / or thalidomide.

41. The kit of any one of claims 38-40, wherein the antiseptic agent comprises chlorhexidine gluconate, alcohol, benzalkonium chloride, and / or hydrogen peroxide.

42. The kit of any one of claims 38-41, wherein the pain reliever comprises menthol, benzocaine, lidocaine, and / or morphine.

43. The kit of any one of claims 38-42, wherein the cancer therapy comprises chemotherapy, immunotherapy, oncolytic virus therapy, and / or hormone therapy.