Mammalian cell populations and pharmaceuticals for cell therapy in mammals, as well as improved cell culture methods.

Abstract

A composition is provided comprising a cell population and pharmaceuticals derived from human gingival fibroblasts, present in proportions not found in natural gingival tissue, but having a cellular phenotype preferentially selected to express proteins favorable for angiogenesis and anti-inflammatory effects, while reducing the formation of cell populations that promote tumorigenesis and / or metalloproteinases that inhibit tissue regeneration. A method for producing such a composition that increases proliferation several times over compared to previously known methods is provided, and a method for using such a composition in a wide range of human cell therapies is also provided.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority based on U.S. Patent Application No. 18 / 301,864, filed on April 17, 2023; U.S. Patent Application No. 18 / 301,856, filed on April 17, 2023; and U.S. Patent Application No. 18 / 301,839, filed on April 17, 2023. This application also claims priority based on U.S. Provisional Patent Application No. 63 / 490,489, filed on March 15, 2023, and European Patent Application No. 23305350.3, filed on March 15, 2023. The entire contents of each of these patent applications are incorporated herein by reference.

[0002] Field of the Invention The present invention generally relates to compositions and medicaments derived from gingival fibroblasts for use in cell therapy, and improved methods for culturing such compositions to create populations of distinct phenotypes that are advantageously different from naturally occurring tissues. In particular, the methods of the present invention are designed to enhance cell proliferation and promote the production of the cell compositions of the present invention in amounts sufficient to enable commercial - scale production. Methods of using the novel compositions for treating various mammalian afflictions (including those affecting epithelial and endothelial surfaces (such as skin aging, pressure ulcers, alopecia, immune disorders, and vascular disorders) and those of osteopathic origin (such as osteoarthritis, cartilage defects, etc.)) are also included.

Background Art

[0003] Background For at least the past 20 years, the use of gingival fibroblasts has been investigated with respect to a wide range of cell - therapy treatments due to the multipotent nature of gingival fibroblasts and their ability to differentiate into a wider range of cells than other types of mesenchymal stem cells. Furthermore, while mesenchymal stem cells are harvested from bone marrow (which poses some risk to the donor), gingival fibroblasts can be easily harvested from gingival tissue, for example, during wisdom - tooth extractions and other dental procedures from unrelated donors.

[0004] The use of gingival fibroblast (GF)-derived compositions, cultured GF cells, and GF-conditioned media is covered by the following U.S. patents and publications: U.S. Patent No. 8,609,085 (Atherosclerosis); U.S. Patent No. 8,303,948 (Skin Wounds); As indicated in U.S. Patent Application No. 2011 / 0097421 (Skin Aging); U.S. Patent No. 10,624,838 (Alopecia); U.S. Patent No. 11,229,670 (Allergic Reactions, Atopic Dermatitis and Asthma); U.S. Patent Application No. 2018 / 0028571 (Cancer); U.S. Patent Application No. 2016 / 0256496 (Human Orthopedic Conditions, Including Osteoarthritis and Cartilage Defects); U.S. Patent Application No. 2020 / 0268805 (Diseases of the Equine Musculoskeletal System, Including Tendinitis, Joint Disorders and Arthropathy) and International Publication No. WO2023 / 007244A1 (Pressure Ulcers), the treatment of many mammalian diseases is described. All of the following patents and applications are licensed or assigned to the assignee of this application and are incorporated herein by reference in their entirety.

[0005] Methods for culturing gingival fibroblasts have been described in numerous previously known publications, including: for example, Hou et al., "Autologous Transplantation of Gingival Fibroblast-Like Cells and a Hydroxylapatite Complex Graft in the Treatment of Periodontal Osseous Defects: Cell Cultivation and Long-Term Report of Cases", Cell Transplantation, 12:787-797 (2003); Ferre et al., "Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells", Stem Cells & Dev., 23(23):2895-2907 (2014); Linard et al., "Therapeutic Potential of Gingival Fibroblasts for Cutaneous Radiation Syndrome: Comparison to Bone Marrow-Mesenchymal Stem Cell Grafts", Stem Cells & Dev., 24(10):1182-1193. (2015) and Ahangar et al., "Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms", npj Regenerative Medicine 5(24)1-10 (2020). All of the aforementioned literature describes methods known prior to culturing cells, including at least the use of culture media (e.g., Eagle's Minimum Essential Medium (EMEM) or Dulbecco's Modified Eagle Medium (DMEM)), antibiotics (e.g., penicillin, streptomycin, or gentamicin), and serum derived from mammalian blood (e.g., 10% fetal bovine serum (FBS)).

[0006] Generally, known methods for culturing gingival fibroblasts involved explanting gingival tissue, followed by either dissection or enzymatic digestion. The cells were then incubated in a humidified incubator with a 5% carbon dioxide atmosphere in the culture medium described above for approximately 2-3 weeks until confluence was achieved, and then trypsinated to produce single-cell suspensions. In some culture methods, the culture medium may be replaced every 72 hours, and after the initial culture period, the gingival fibroblast colonies may be trypsinated again to produce single-cell suspensions, which are then seeded in fresh medium for a further 2 weeks (this is often called "passage"). After the final passage and trypsination steps, the cultured cells may be washed and collected for use in the desired application.

[0007] Regarding previously known culture methods and the resulting cell compositions, many drawbacks exist. One such drawback is the time required to culture a small number of gingival fibroblasts to produce cell colonies large enough to support commercial applications. In particular, it is desirable to provide cell compositions and methods for culturing such compositions to enhance proliferation during the culture stage. In this way, a sufficient cell population for a commercialized method to treat the disease condition described in the patent cited above can be realized, thereby helping to move the use of gingival fibroblasts from a research-oriented technique to a practical treatment application on a commercial scale.

[0008] Another drawback of previously known compositions and methods is the inability to tailor the protein expression characteristics of the cell phenotype in a cultured cell population to a specific purpose. For example, it is known that native gingival fibroblasts cultured in vitro contain a large cell population that expresses CD146 (also known as the melanoma cell adhesion molecule and associated with the tumorigenicity of melanoma cells). Therefore, for many of the treatments described in the patents and applications referenced above, it is desirable to restrict the phenotype to one that expresses CD146. Similarly, it is desirable to culture gingival fibroblasts to enhance the expression of certain phenotypes (e.g., CD90 (also known as THY1) and TIMP1). Of these phenotypes, TIMP1 inhibits the production of matrix metalloproteinases and reduces inflammatory responses. In view of the foregoing, it is desirable to provide compositions and pharmaceuticals derived from gingival fibroblasts, which include a population of cells cultured to have a specific protein expression phenotype, wherein the phenotype is preferentially selected to express proteins favorable for angiogenesis and anti-inflammatory effects, while reducing the formation of cell populations that promote tumorigenesis and / or metalloproteinases that inhibit tissue regeneration. It is even more desirable to provide compositions, pharmaceuticals, and methods for culturing such compositions in sufficient quantities to facilitate the use of such compositions in the treatment of various mammalian diseases (including, but not limited to, atherosclerosis, skin wounds, skin aging, alopecia, allergic reactions, atopic dermatitis, asthma, pressure ulcers, cancer, and orthopedic conditions (including osteoarthritis and cartilage defects, tendinitis, joint disorders and arthropathy)). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent No. 8,609,085 [Patent Document 2] U.S. Patent No. 8,303,948 [License 3] Details of U.S. Patent and Trademark Office No. 2011 / 0097421 [License 4] U.S. Patent No. 10,624,838 [Patent Document 5] U.S. Patent No. 11,229,670 [License 6] Details of U.S. Patent and Trademark Office No. 2018 / 0028571 [License 7] Details of U.S. Patent and Trademark Office No. 2016 / 0256496 [License 8] Details of U.S. Patent and Trademark Office No. 2020 / 0268805 [License 9] International Gazette No. 2023 / 007244A [Non-licensed literature]

[0010] [Non-licensed Document 1] Hou et al., "Autologous Transplantation of Gingival Fibroblast-Like Cells and a Hydroxylapatite Complex Graft in the Treatment of Periodontal Osseous Defects: Cell Cultivation and Long-Term Report of Cases", Cell Transplantation, 12:787-797 (2003) [Non-licensed Document 2] Ferre et al., "Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells", Stem Cells & Dev., 23(23):2895-2907 (2014) [Non-licensed Document 3] Linard et al., “Therapeutic Potential of Gingival Fibroblasts for Cutaneous Radiation Syndrome: Comparison to Bone Marrow-Mesenchymal Stem Cell Grafts”, Stem Cells & Dev., 24(10):1182-1193 (2015) [Non-Patent Document 4] Ahangar et al., “Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms”, npj Regenerative Medicine 5(24)1-10 (2020) [Overview of the Initiative]

[0011] Summary of the Invention The present invention relates to compositions, pharmaceuticals, and methods for producing compositions of gingival fibroblasts having a phenotype cultured to express preferred proteins. In particular, the gingival fibroblast cell population of the present invention differs from gingival fibroblasts present in that its cellular phenotype is preferentially selected to express proteins favorable for angiogenesis and anti-inflammatory effects, while reducing the formation of cell populations that promote tumorigenicity and / or metalloproteinases that inhibit tissue regeneration. In one embodiment, the composition differs from a population of natural gingival fibroblasts by having a cell population or conditioned medium in which at least about 90% of the cells have a phenotype expressing THY1, CD99, or CD63, and about 20% or less of the cells have a phenotype expressing CD146.

[0012] According to a further aspect of the present invention, there is provided a method for enhancing cell growth to realistically produce commercial scale amounts of the composition having the above-specified phenotypic characteristics for culturing the composition. In particular, the method of the present invention advantageously not only allows for a preferential selection of cell phenotypes but also permits culturing at a substantially increased rate of cell growth. In one preferred embodiment, the cell culture process includes multiple passages of the cells in 20% FBS with non-essential amino acids and frequent culture medium changes, resulting in more than a doubling of cell growth compared to prior art culture methods.

[0013] According to the present invention further, methods of using the compositions and medicaments of the present invention are provided for treating a number of mammalian pathologies (including atherosclerosis, skin wounds, skin aging, alopecia, allergic reactions, atopic dermatitis, asthma, cancer, human orthopedic pathologies (including osteoarthritis and cartilage defects, tendonitis, joint disorders and arthrosis) and pressure ulcers). The cultured gingival fibroblasts and conditioned media of the present invention may also be useful in regenerating excised or damaged nerves, such as spinal cord injuries. Based on preliminary animal data, the response to the aforementioned treatments may be dose-dependent, and as a result, in order to obtain a clinically significant and sustained treatment response, it has been observed that the treatment should deliver a cell population of between 5 million and 40 million cells, and more preferably, approximately 20 million cells, to the treatment site.

[0014] The composition of the present invention may include a cultured mammalian cell population as defined above in a liquid cell culture medium, or a medicament derived from a cultured mammalian cell population as defined above, or a pharmaceutical or cosmetic composition including a cultured mammalian cell population as defined above and at least one pharmaceutically or cosmetically acceptable carrier or excipient.

[0015] Other features of the systems and methods of the present invention will be apparent upon reference to the following detailed description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Brief Description of the Drawings [Figure 1] Figure 1 is a table showing the evolution of the gingival fibroblast phenotype in a cell population cultured during the fifth passage of culturing according to the principles of the present invention.

[0017] [Figure 2] Figure 2 is a table showing representative protein expression characteristics and growth indices for various conventional culture media as compared to those of the present invention.

[0018] [Figure 3] Figure 3 is a chart showing the effect of using different concentrations of FBS during cell culture and changing the culture medium during the first passage of cell culture.

[0019] [Figure 4] Figure 4 is a chart showing the effect of changing the culture medium during the second passage of cell culture.

[0020] [Figure 5] Figure 5 is a chart showing the effect on the cell population phenotype resulting from culturing gingival fibroblasts with and without non-essential amino acids during further culture passages.

[0021] [Figure 6] Figure 6 is a series of bar graphs showing data achieved in a dog model regarding the treatment day and one-year follow-up, conducted with different doses of the composition of the present invention.

[0022] [Figure 7] Figure 7 is a series of bar graphs showing the percentage of effectiveness of the treatment conducted as shown in Figure 6.

[0023] [Figure 8]Figure 8 is a series of bar graphs showing the dose-dependence and persistence of the treatment performed as shown in Figure 6, as a function of the number of months since the treatment date.

[0024] [Figure 9] Figure 9 is a table summarizing the results obtained in the first group of horses 90 days after injection of a composition according to the present invention for treating fetlock joint disorders.

[0025] [Figure 10] Figure 10 is a table summarizing the results of tenderness (sensitivity) data obtained in the second group of horses 90 days after injection of a composition according to the present invention for treating tendon disorders.

[0026] [Figure 11] Figure 11 is a graph showing a summary of the joint deformation data observed in the second group of horses described above, 90 days after injection of a composition according to the present invention to treat tendon disorders.

[0027] [Figure 12] Figure 12 is a series of bar graphs showing the improvement in health scores in the second group of horses as a function of the number of days since the treatment date. [Modes for carrying out the invention]

[0028] Detailed description of the invention The present invention relates to compositions and pharmaceuticals comprising a population of cells derived from gingival fibroblasts having a phenotype occurring at a rate not found in natural gingival tissue. The present invention further provides methods for producing such compositions, as well as methods for using such compositions and conditioned media in a wide range of mammalian cell therapies, including humans, dogs, horses, and cats.

[0029] Compositions and pharmaceuticals of the present invention Compositions according to the principles of the present invention preferably comprise a cell population that expresses angiogenic and anti-inflammatory proteins, and has a low proportion of cells with a phenotype that expresses tumorigenicity-associated proteins or promotes the formation of metalloproteinases. The cell population is cultured from gingival fibroblasts (as described below) to have phenotypic characteristics different from those that occur in natural gingival tissue. The cell population of the present invention may be packaged directly for pharmaceutical use or may be used to create conditioned media for pharmaceutical use.

[0030] In a preferred embodiment, the composition comprises about 10% or less of the cells of the cultured mammalian cell population expressing at least one mRNA selected from the group consisting of the mRNAs of CD146 (also known as melanoma cell adhesion molecule (MCAM)), VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IFI44L, RSAD2, IFIT1, IFNA1, and IFNG.

[0031] Preferably, at least about 50% of the cells in the cultured mammalian cell population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs, and more preferably, at least about 90% of the cells in the cultured mammalian cell population express CD63 mRNA and TIMP1 mRNA. TIMP1 represents TIMP metallopeptidase inhibitor 1.

[0032] In laboratory studies, the applicants have observed that the protein expression characteristics (as measured by mRNA transcripts) of cultured gingival fibroblasts according to the present invention for CD63 and TIMP1 are substantially different from those of these proteins when produced by natural gingival tissue. The present invention's method of culturing gingival fibroblasts over several culture passages, as described below, preferentially enhances desirable phenotypes while limiting the growth of less desirable phenotypes.

[0033] Figure 1 is a chart showing the evaluation of specific cell protein expression phenotypes cultured from human gingival fibroblasts over multiple passages to generate a cell population according to the present invention. In particular, the phenotypic proportions of naturally occurring gingival tissue include a population in which only 30.2% of cells express the desirable CD90 protein, 6.9% express the desirable CD63, while 20.7% express the less desirable CD146 protein (melanoma cell adhesion molecule). However, as shown in subsequent culture passages 0-5, the proportion of desirable phenotypes is preferentially increased, while the proliferation of undesirable phenotypes is limited. For example, by the completion of passage 2, the proportion of cells expressing all CD90, CD105, CD73, and CD63 proteins exceeds 95%, while the proportion of cells expressing CD146 is 13.4%. However, during the next second passage, the proportion of cells expressing CD146 is reduced to 6.2%, more than half, without significantly reducing the proportion of cells expressing CD90, CD105, CD73, and CD63 proteins.

[0034] Preferably, essentially all cells in the cultured mammalian cell population have an undifferentiated phenotype, more preferably a fibroblast-like phenotype. The cultured mammalian cell population preferably arises from mucosa, more preferably from oral mucosa, particularly from the mucosa of the cheek, tongue, palate, lip, sublingual, or gingiva. Procedures for taking samples or biopsies from mucosa are well known to those skilled in the art. Generally, cells from a biopsy are separated by enzymatic digestion before being magnified.

[0035] A suitable cultured mammalian cell population may be derived from any mammal, for example, livestock (selected from the group consisting of horses (especially horses), camels (especially camels, dromedaries, or llamas), cattle, goats, dogs (especially canines), cats (especially felines), and weasels). For compositions intended for use in humans, it is preferable that the cultured mammalian cell population is obtained from a human cell population. The cell population according to the present invention may be autologous or heterologous. As those skilled in the art will understand, the individual from which the cells are collected and the individual to which the cells are administered are preferably of the same species, but they may also be of different species.

[0036] The above-mentioned cell populations may exist in various forms (e.g., suspensions in liquid (e.g., physiological solutions, PBS, or cell culture media), cultures on solid culture media, or pellets (especially centrifuged pellets)).

[0037] When used herein, a composition or pharmaceutical according to the present invention contains a population of cells according to the present invention in an amount effective for prevention or treatment. Preferably, a composition according to the present invention contains 5 million to 40 million cells according to the present invention, more preferably 10 million to 30 million cells according to the present invention, even more preferably 15 million to 25 million cells according to the present invention, and most preferably about 20 million cells according to the present invention. The quantification of cells according to the present invention may be performed by any method for counting mammalian cells known to those skilled in the art (e.g., a cell counter).

[0038] The above composition or pharmaceutical may be in any form suitable for its intended application. In a preferred embodiment of the present invention, the composition may be in an injectable form suitable for delivery, for example, by injection using a syringe. In an alternative embodiment, the composition may be in a form suitable for topical administration. In that case, the composition may preferably be in the form of a lotion, cream, ointment, gel, spray, wipe, bandage, pad or patch.

[0039] As used herein, the “conditioned media” product may be any product derived from and obtained from the mammalian cell population of the present invention, or containing secretions from the cell population. Preferably, the conditioned media product derived from the cultured mammalian cell population is a cell extract. The extract may be obtained by any cell fragmentation method known in the art, including the group consisting of membrane extracts, cytoplasmic extracts, or nuclear extracts.

[0040] The conditioned medium according to the present invention may further include a liquid cell culture medium in which the cells of the cell population of the present invention are in contact for a sufficient amount of time, in particular, for the cells to secrete into the medium. Thus, the conditioned medium according to the present invention includes secretions from the cells of the cultured mammalian cell population according to the present invention. Preferably, the cells are in contact with the culture medium for at least 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, or 48 hours, and generally less than 56 hours or 72 hours.

[0041] The conditioned medium according to the present invention can be subjected to processing steps (e.g., centrifugation, filtration, or concentration). In particular, the conditioned medium according to the present invention may be a concentrated conditioned medium, or more specifically, a concentrated medium obtained by concentrating the concentrated conditioned medium 2-fold, 5-fold, 10-fold, 25-fold, or 50-fold relative to the unconcentrated conditioned medium from which the concentrated conditioned medium is derived.

[0042] Cells, cell populations, phenotypes, etc., that express or do not express the indicated protein or mRNA are described herein. According to this disclosure, the indicated cells are considered not to express the indicated protein or mRNA if standard experimental assays (e.g., single-nuclear RNA sequencing (snRNAseq), fluorescence-activated cell sorting (FACS), etc., as substantially described by Grindberg et al., Proc. Natl Acad. Sci. USA 110:19802-19807 (2013)) cannot detect a recognizable level of the indicated protein or mRNA. Conversely, cells are considered to express the indicated protein or mRNA if the above methods can detect a recognizable level of the indicated protein or mRNA.

[0043] As will be recognized by those skilled in the art, protein expression can be measured in accordance with this disclosure by measuring the corresponding mRNA transcript.

[0044] Culture of a preferred cell population According to one aspect of the present invention, gingival fibroblasts are cultured through 5 to 7 passages after extraction from natural gingival tissue in order to preferentially regulate their phenotype in the composition, thereby increasing desirable protein expression characteristics while suppressing undesirable ones.

[0045] In one embodiment, heterologous mammalian gingival tissue is collected from the oral cavity of a suitable mammal for the species in which the cell composition to be cultured, or the conditioned medium, will ultimately be used. The collected tissue is cut into small fragments with a surgical scalpel, and these fragments are digested by enzymes. The dissociated cells are washed in phosphate-buffered saline (PBS), and a cell suspension is created by placing the washed cells in a mixture of culture medium and 20% FBS. By example, suitable culture media according to the present invention include Eagle Minimum Essential Medium (MEM or EMEM), Eagle Minimum Essential Medium Alpha Modified (Alpha MEM), and Eagle Basal Medium (BME).

[0046] Next, 10,000 cells / cm 2 Zero-passaging is performed at the cell seeding density. Here, the cells are cultured in a culture flask containing culture medium, 20% FBS, antibiotics, and basic fibroblast growth factor (bFGF). Basic fibroblast growth factor (bFGF) refers to recombinant human bFGF produced in Escherichia coli having human bFGF, and more preferably the sequence identified by Sequence ID No. 1 (listed herein by reference). The medium is removed, and the cells are washed with PBS on days 3 and 10. On day 21, the medium is removed, the cells are washed again with PBS, trypsinized, and removed from the flask. By the completion of zero-passaging after 21 days, the gingival fibroblast population is generally expanded 6-fold.

[0047] During the first passaging, the cells in a mixture of culture medium, 20% FBS, and bFGF were kept at 37°C in an atmosphere of 95% oxygen and 5% carbon dioxide, 95% humidity, at a density of 4,500 cells / cm³. 2 The cells are cultured in a culture flask at a cell seeding density. On day 3, the cells are washed with PBS and provided with a fresh mixture of culture medium, 20% FBS, and bFGF. The cells are cultured for a further 3 days, after which they are again trypsinized. During the first 6-day passage, the cell population generally expands about 15-fold, for example, from about 10 million cells to about 143 million cells in the exemplary embodiment. After further washing with PBS, the cells can be placed in storage culture medium consisting of a solution of approximately human serum albumin 4% w / v (USP / Ph.Eur) and 10% DMSO (USP / Ph.Eur) and stored in quantities of 10 million cells / storage tube.

[0048] In the second passaging, the cells from one storage tube were placed in a mixture of culture medium, 20% FBS, and bFGF at a rate of 4,500 cells / cm³. 2The cells are dispersed at a seeding density, which is renewed after 4 days. On day 7, the culture medium mixture is removed. Upon completion of passage 2, the cells are generally, again, magnified by approximately 15 times.

[0049] In the third passage, the cell density was 4,500 cells / cm³. 2 The cells are seeded at a density, for example, in a multi-plate bioreactor or other similar device for large-scale expansion, with a mixture of culture medium, 20% FBS, and bFGF. The culture medium mixture is replaced with a fresh mixture after 3 days and cultured for a further 3 days. After 6 days, the cells are trypsinized, rinsed in PBS, and placed in storage tubes with a storage medium consisting of a solution of approximately human serum albumin 4% w / v (USP / Ph.Eur) and 10% DMSO (USP / Ph.Eur). Upon completion of passage 3, the cells have generally expanded about 15-fold. Preferably, the cells can be distributed for storage into tubes, each holding about 10 million cells.

[0050] In the fourth passage, the cells from one storage tube were placed in a mixture of culture medium, 20% FBS, and bFGF at a rate of 4,500 cells / cm³. 2 The cells are dispersed at a seeding density, which is refreshed after 4 days, washed with PBS, and placed in the bioreactor for an additional 6 days for the fifth passage, according to the second passage protocol. Upon completion of the fourth passage, the cells have generally expanded by approximately 15-fold, and are trypsinized, washed with PBS, and prepared for further culture in the fifth passage.

[0051] In the fifth passage, the cell density was 4,500 cells / cm³. 2At a density, the cells are seeded in a bioreactor or other large-scale expansion device with a mixture of culture medium, 20% FBS, and bFGF, which is replaced with a fresh mixture of culture medium after 3 days. On day 3, the culture medium mixture is removed and replaced with a fresh culture medium mixture, omitting fetal bovine serum and supplemented with interleukin-1β (IL-1β). After 1 day, the culture medium mixture is removed, the cells are generally magnified about 15 times, trypsinized, rinsed with PBS, and stored in storage tubes with the storage medium as described above, each holding 10 million cells.

[0052] Preferably, as described above, the cell population of the present invention is cultured for a fifth passage in the presence of an inflammatory cytokine (e.g., IL-1β) before being used in a therapeutic or cosmetic application according to the present invention. The concentration of the inflammatory cytokine (particularly IL-1β) in the culture medium may be 0.1 ng / ml to 10 ng / ml, more preferably 0.5 ng / ml to 2 ng / ml. The inflammatory cytokine (particularly IL-1β) may be recovered from the cell culture, for example, by washing the cells, before being used in a therapeutic or cosmetic application according to the present invention.

[0053] The cell culture of the present invention can be concentrated, for example, by filtration or centrifugation. In particular, the cell culture can be concentrated 2-fold, 5-fold, 10-fold, 25-fold, or 50-fold relative to the unconcentrated cell culture from which it is derived.

[0054] Culture of a composition according to the present invention by the process described above yields an exemplary cell population having the protein expression characteristics shown in Figure 2. While not intended to be limiting, a key difference in the process of culturing gingival fibroblasts according to the present invention compared to culture methods described in previously known methods is the use of 20% FBS and basic fibroblast growth factor during cell culture, and the replacement of the culture medium mixture at predetermined intervals during multiple 6-7 day passages. Furthermore, the method further includes the use of IL-1β and / or non-essential amino acids during the final passage to modulate the protein expression characteristics of the target cell population, as shown in Figure 1.

[0055] Referring here to Figure 2, another important aspect of the present invention is the ability of the above process to enhance cell proliferation during the culture process. This index (also called the expansion index) indicates the degree of magnification by which the cells are expanding during the specified passage. Compared to the cell culture methods described in the above literature, the method of the present invention has been observed to achieve significantly greater proliferation. This, in turn, makes it possible to produce the composition of the present invention in sufficient quantities to support commercial application within the specified period. Specifically, Figure 2 reports the results achieved after five culture passages using a conventional culture medium containing 10% FBS and Scarcell medium. An exemplary formulation of Scarcell medium is DMEM, 20% FBS, and bFGF, as shown above. While not intended to limit the method of action, it is assumed that the stability of the above cell culture is maintained by using higher concentrations of fetal bovine serum and frequently changing it.

[0056] Figure 2 shows that for conventional media consisting of various combinations of DMEM, 3% Lysat, heparin, and 10% FBS, with and without non-essential amino acids, the growth index varied from approximately 5 to 9 times. By comparison, the results achieved with Scarcell medium were up to 17 times for one source of gingival fibroblasts and up to 22 times for the other source. Furthermore, the method according to the present invention enhances the yield of cells preferably expressing CD63 protein between approximately 20 and 50%, as described above. These results demonstrate a 2-3 times greater ability to expand gingival fibroblasts during culture and thus provide a pathway to a larger supply of the composition of the present invention during the specified period, and potentially a reduction in the cost for such production.

[0057] Referring to Figures 3 and 4, graphs are provided showing the increase in the gingival fibroblast population as a function of time and culture medium. In particular, the lines drawn by white and black squares in Figure 3 show the progress of cell volume cultured during the first passage in medium with 10% FBS. The line drawn by black triangles corresponds to culture where the culture medium was changed after the first four days, while the line drawn by white squares corresponds to culture without medium change. In Figure 3, the lines drawn by black diamonds and black squares show the progress of cell volume cultured during the first passage in medium with 20% FBS. Here, the medium was either changed after the first four days (black square) or not (black diamond). All four lines in Figure 3 show that the viable cell volume stagnates or decreases after 7 days of culture in 10% FBS medium (with medium change), providing approximately the same cell count as achieved using 20% ​​FBS (without change). However, unexpectedly, using 20% ​​FBS while changing the medium after the first four days similarly resulted in more than a doubling of the number of viable cells after 7 days of culture.

[0058] Figure 4 provides results similar to those shown in Figure 3, except that the second culture passage was performed in a culture medium containing 20% ​​FBS. Specifically, in Figure 4, the lines drawn with black diamonds represent the medium containing 20% ​​FBS (where the medium was not changed after 4 days), and the lines drawn with black squares represent the growth in cell volume cultured in the medium containing 20% ​​FBS (where the medium was changed after the first 4 days). As shown in Figure 4, the number of viable cells begins to decrease after 6 days when the medium is not changed, and begins to plateau after 7 days when the medium is changed. Notably, and unexpectedly, changing the medium after the first 4 days during the second culture passage approximately doubles the yield of viable cells during the second passage.

[0059] Now, looking to Figure 5, an alternative execution of the process according to the present invention is described. Here, one or more further culture passages are completed to reduce the cell population expressing the undesirable protein CD146. In this method, cells completing the fourth passage (as described above) are cultured for the fifth, sixth, and optional seventh passages (substantially as described above), except that non-essential amino acids are added to the culture medium. As shown in Figure 5, the percentage of cells expressing the CD146 protein increases from 27.9% at the completion of the fourth passage to 52.7% in the fifth passage, and then to 14.2% in the sixth passage when cultured with NEAA. By comparison, omitting NEAA from the culture medium during the fifth passage increases the percentage of cells expressing CD146 to 74.4%, which is reduced to just 25.8% after two further passages. Therefore, the method of the present invention specifically includes adding NEAA to the culture medium during passaging in the later stages of the cell culture process, preferably, in order to adjust the proportion of the above-mentioned cell population and change the protein expression characteristics.

[0060] Therapeutic use of the composition of the present invention Cell cultures and conditioned media prepared in accordance with the principles of the present invention can, advantageously, be used to treat a wide range of mammalian diseases, as described in the patents and publications incorporated herein and assigned or licensed to the assignees of the present invention. For example, cultured cell suspensions or conditioned media products derived therefrom can be used to treat atherosclerosis, skin wounds, skin aging, alopecia, allergic reactions, atopic dermatitis, asthma, cancer, human orthopedic conditions (including osteoarthritis and cartilage defects, tendinitis, joint disorders and arthropathy), and pressure ulcers. The compositions and conditioned media of the present invention can also be used to regenerate nerve tissue, for example, to treat spinal cord injury. Administration of cultured mammalian cell populations as defined above or conditioned media products derived therefrom, or pharmaceutical compositions derived therefrom, can be carried out by any method known in the art, including administration subcutaneously, intravenously, intramuscularly, intradermally, or topically, near or in the area of ​​the skin to be treated.

[0061] For example, skin-related disorders may be treated by topical application of the above composition or conditioned medium, while vascular diseases may be treated by intravascular injection or delivery of other devices having a stent or reservoir (from which the above composition may be delivered). Further applications, such as treatment of bone disorders, may involve direct injection near a joint or tendon.

[0062] Surgical wounds are wounds that occur during surgical procedures. Notably, such surgical wounds include wounds that occur in the course of plastic surgery and reconstructive surgery or scar formation (e.g., hypertrophic scars). The plastic surgery and reconstructive surgery procedures mentioned above may be of any type, e.g., breast surgery, abdominal surgery, nasal surgery, ear surgery, or removal of skin defects. As intended herein, skin defects are related to abnormal skin formation found in individuals with a genetic predisposition or as a result of abnormal skin development during embryogenesis, and notably include giant nevi, cleft lip, and keloids.

[0063] As intended herein, “treating a skin wound” relates to promoting, accelerating, or improving healing at the site of the wound, i.e., the formation of functional skin at the site of the wound. Where intended herein, “functional skin” refers to skin that has restored its mechanical properties and barrier function, in particular, with respect to an uninjured area of ​​skin. Inflammatory skin lesions generally include inflammatory skin diseases, particularly those resulting from chronic inflammatory skin diseases, and may include the group consisting of dermatitis, inflammatory rashes, ichthyosis, and psoriasis. Chronic inflammatory skin diseases also include rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0064] The cultured cell suspensions or conditioned medium products derived therefrom may also find applications in cosmetic use. Where intended herein, “cosmetically acceptable carrier or excipient” refers to any substance suitable for cosmetic compositions. Preferably, the cosmetically acceptable carrier or excipient is suitable for topical application. Cosmetically acceptable carriers or excipients according to the present invention include, but are not limited to, any of the standard cosmetic carriers or excipients known to those skilled in the art, such as water, vegetable oils, mineral oils, fatty acid alcohols, and natural waxes. [Examples]

[0065] Example 1 Single nuclear RNA sequencing of the cell population according to the present invention Single-nuclear RNA sequencing (snRNAseq) was performed on the cell population of the present invention prepared according to the method described above. The snRNAseq method was originally described in Grindberg et al., Proc. Natl Acad. Sci. USA 110:19802-19807 (2013) and is known to those skilled in the art. The results of the analysis are shown in the table below. [Table 1-1] [Table 1-2]

[0066] Example 2 Protein expressed by the cell population of the present invention The expression of a specific protein was determined by fluorescence-activated cell sorting (FACS), as shown in the table below. [Table 2]

[0067] Results of animal research Initial tests of compositions produced according to the method of the present invention were conducted in canine and equine animal models, specifically including the treatment of osteoarthritis in a group of 95 dogs, and the treatment of separate groups of horses suffering from fetlock joint disorders and tendon disorders. A summary of the results achieved during these tests is discussed in relation to Figures 6-12.

[0068] Treatment of secondary osteoarthritis in dogs Degenerative osteoarthritis is a frequent and well-known ailment in canine veterinary medicine. This disease is often triggered by damage to the subcostal soft tissue, articular cartilage, or a combination of both. This trigger leads to degeneration of the articular cartilage, involving bone and tissue changes. The specific origin of the lesion, such as inflammation of the joint or synovial surface, or the disease leading to joint degeneration, is not a determinant of the outcome. Instead, synovitis leads to the production of pro-inflammatory cytokines and metalloproteinases, which contribute to the chronic development of the disease. Furthermore, synovitis causes joint exudates, which contribute to joint pain and joint instability. The prevalence in dogs varies between 2% and 20% at one year of age. Osteoarthritis can lead to significant complications, particularly in association with obesity, requiring long-term treatment through anti-inflammatory drugs or prosthetic joint replacement surgery.

[0069] Current treatments for canine osteoarthritis do not have a positive effect on cartilage regeneration and primarily target symptoms, such as reducing pain caused by joint inflammation. Research into alternative treatments has recently led to the development of cell-based therapies, mainly in light of the potential regenerative potential of such treatments. Current allogeneic cell therapies are limited by their half-life after administration to the subject. The aim of this study was to evaluate the safety and efficacy of cell therapy based on the use of allogeneic gingival fibroblast cultures in pet dogs suffering from osteoarthritis.

[0070] A randomized study of 95 dogs was conducted using a single intra-articular injection to study the efficacy, safety, and tolerability of canine gingival fibroblasts in the treatment of osteoarthritis. The injection vehicle consisted of 10% DMSO, 88% DMEM, 1% L-glutamine, and 1% non-essential amino acids per 1 mL total volume. Sixteen dogs received injections of the injection vehicle without gingival fibroblasts (placebo), 28 dogs received injections of 2 million cultured cells, 28 dogs received injections of 5 million cultured cells, and 23 dogs received injections of 10 million cultured cells. The study parameters were as follows: Inclusion criteria were dogs with one or more moderate to severe osteoarthritis resistant to standard treatment; exclusion criteria included sarcoma, prior cell therapy, surgery, or prior multiple injections.

[0071] The dogs described above were evaluated by a veterinarian one month after injection and by their owners 12 months after injection. Lameness and pain were scored according to the following rules: [Table 3]

[0072] More specifically, clinical effectiveness was assessed using the Canine Brief Pain Inventory (CBPI) questionnaire as described in Brown et al., "Ability of the Canine Brief Pain Inventory to detect response to treatment in dogs with osteoarthritis," J. Am. Vet. Med. Assoc., 233(8):1278-1283 (2008), including evaluation of pain intensity scores (CBPI questions 1-4), pain interference scores (CBPI questions 5-10), and the overall CBPI score. Clinical effectiveness was assessed at 1 month and 12 months. Intermediate time points (3 months, 6 months, and 9 months) were determined using the Kaplan-Meier methodology (dogs whose effectiveness was observed up to 6 months were counted at the 3-month and 6-month time points).

[0073] The results of the study are discussed in relation to Figures 6-8. One month after injection, claudication improved in all groups (Figure 6), but there was no improvement in the placebo group (23% reduction). The groups with 2 million, 5 million, and 10 million cells showed reductions of 52%, 53%, and 41%, respectively (Figure 7). All doses showed a significant benefit over placebo at one month.

[0074] One month after injection, pain improved by 37%, 49%, 41%, and 45% in all groups for the placebo, 2 million, 5 million, and 10 million cell groups, respectively (Figures 6 and 7). No statistical significance regarding pain was observed among the 2 million, 5 million, and 10 million cell groups. The placebo group showed transient efficacy. This may be explained by the fact that the joint was washed with a saline solution before injecting the cells to remove debris and thereby reduce inflammation. The efficacy was lower than that obtained with gingival fibroblast injection.

[0075] No improvement was observed with any of the treatments for any of the other clinical parameters observed before injection and one month after injection, namely muscle condition, joint exudate, and crackles (Figures 6 and 7). However, with respect to all parameters, cell therapy was significantly effective before and after treatment for all doses used; the sex of the animals did not affect the effectiveness of the treatment. No adverse events were observed during the first 72 hours. At one month, the effectiveness in all groups was not affected by the dog's weight, age, or initial lameness, and no correlation was observed between the dog's initial clinical condition before injection and its age or weight.

[0076] Clinical efficacy was assessed one year after injection based on the CBPI questionnaire (completed by the dog's owner) and interviews. The mean duration of efficacy was 2.6 months, 5.5 months, 6.3 months, and 9.1 months for the placebo, 2 million, 5 million, and 10 million cell groups, respectively. All cultured cell groups differed significantly from the placebo group, with the 10 million cell group showing significantly longer efficacy than the 2 million and 5 million cell groups. The percentage of dogs observing efficacy for 12 months after injection was 12.5%, 25%, 40%, and 70% for the placebo, 2 million, 5 million, and 10 million cell groups, respectively, as shown in Figure 8. All cell groups showed a significant difference compared to the placebo group at 12 months, and the 10 million cell group showed a significant difference compared to the 2 million and 5 million cell groups at 12 months.

[0077] Arthroscopy was performed on one dog before injection and 4 months after injection. The dog had a non-smooth cartilage margin with loss of ultrastructure of the joint (i.e., no clear gaps, and a mixture of inflammatory cells and chondrocytes). 4 months after injection, arthroscopy revealed smooth cartilage with normal ultrastructure (i.e., chondrocytes in clear lumens). This case confirms the clinical findings of joint repair after intra-articular injection of cultured cells prepared according to the present invention.

[0078] Canine dose-response studies showed that intra-articular injections of 2 million, 5 million, and 10 million cells were more effective than placebo in treating dogs with osteoarthritis one month and 12 months after the injection. The effect on lameness one month after injection was similar for all three doses at 12 months, but the 5 million and 10 million cell doses were statistically different from placebo, while the 2 million cell dose was not. The more than 50% success observed at one year can be hypothesized to be related to cartilage repair.

[0079] The variability of subjects in the study (which was conducted in an uncontrolled environment) is similar to that of human populations, suggesting the potential transfer of preclinical study results to humans. Notably, the above study was conducted in a cohort of dogs that were very different in terms of breed, genetic background, sex, and weight / size. The sustained clinical efficacy in a heterogeneous canine population over one year demonstrates the remarkable potential and robustness of the composition of the present invention for the treatment of osteoarthritis of the knee.

[0080] Treatment of equine tendon disorders The objective of this study was to evaluate the tolerability and efficacy of local injection of gingival fibroblasts in the treatment of acute tendon injuries of the deep flexor digitorum tendons in 10 competition horses, including mares and geldings, racehorses, and eventing horses. The study involved a single 2.5 mL intratendinous injection of 20 million cultured equine gingival fibroblasts in appropriate culture medium. Clinical examinations were performed immediately before injection and 90 days later. Scoring for clinical evaluation included deformity, sensitivity, and fever, each on a scale of 1–3 (3 being the worst), as well as dynamic examination of lameness on a scale of 1–5 (5 being the worst). Ultrasonography of the injured limb was also performed to determine hypo-echogenicity (5 indicates high echogenicity and severe inflammation).

[0081] A single injection administered by syringe consisted of: 2.5 mL of 1% sodium hyaluronate solution, 25 mg of sodium chloride, disodium phosphate, monosodium phosphate, water, 20 million cultured equine gingival fibroblasts as described above, 200 μL of pure conditioned medium, and parasecretion of gingival fibroblasts over 24 hours. The composition of 500 mL of pure conditioned medium was L-glutamic acid, 2400 mg / L sodium bicarbonate, buffering agent (HEPES), sodium pyruvate, hypoxanthine, thymidine, trace elements, growth factors, and 1.1 mg / L phenol red. Heterogeneous gingival fibroblasts in the suspension were obtained from gingival biopsies of foals at birth from volunteer owners.

[0082] The results of the study are shown in Figure 9, along with a detailed legend explaining the scoring system. In particular, as shown in Figure 9, at 3 months, ultrasound evolution showed complete disappearance of the hypoechoic lesion areas initially observed by ultrasound before injection in five horses (EC1_2, EC1_4, EC1_5, EC1_8, and EC1_10). Four horses (EC1_1, EC1_3, EC1_7, and EC1_9) showed persistence of some hypoechoic lesions; one horse (EC1_6) clearly showed persistence of hypoechoic lesions. The favorable evolution of tendon lesions observed by ultrasound at 3 months post-injection persisted at re-examination at 6 months.

[0083] Ultrasound and clinical progress were evaluated beyond 6 months: Six horses showed a recurrence of tendinitis between 7 and 11 months after injection; two horses were selected for other pathological causes; and one horse (eventing, EC1_7) returned to normal activity. All horses running in controlled gallops showed a recurrence of tendinitis within 12 months. One racehorse (EC1_8) showed favorable progress in its tendinitis at 12 months, competing in 8 races after recovery, achieving 3 wins and 4th place. Furthermore, although the deformity observed before injection persisted at the 90-day evaluation, ultrasound examination showed significant filling of the lesion, and none of the horses showed lameness, with only very few showing heat and tenderness.

[0084] This clinical trial demonstrated that a gingival fibroblast composition cultured according to the principles of the present invention has a good ability to resolve tendon disorders in horses.

[0085] Treatment of horse fetlock joint disorders The purpose of this study was to evaluate the tolerability and efficacy of local injection of equine gingival fibroblasts cultured according to the principles of the present invention in the treatment of fetlock joint disorders in racing horses. Ten horses participated in this trial. The fetlock is the joint where the cannon bone, proximal sesamoid bone, and first phalanx come into contact.

[0086] The above study was conducted as a single 2.5 mL intra-articular injection of 20 million cultured gingival cells in appropriate culture medium, with examinations performed on days 3, 10, and 90. The injection of 20 million cultured cells was prepared as described above for tendon injury studies. Inclusion criteria were clinical examinations to assess passive flexion, degree of lameness, joint deformity, and tenderness. On days 3, 10, and 90, horses were examined for passive flexion, degree of lameness, joint deformity, and tenderness (sensitivity). Favorable outcomes included return to activity, return to training, and return to competition.

[0087] A group of 10 horses of varying ages and sexes participated in the study. The results of the study are shown in Figures 10-12. As shown in Figures 10 and 12, many horses experienced a 100% reduction in sensitivity by day 10, which persisted until day 90, while other horses continued to improve sensitivity throughout the observation period. Similarly, as shown in Figures 11 and 12, some horses experienced a reduction in joint deformation by day 3, which stabilized and persisted until day 90, while other horses showed continued improvement throughout the observation period. The rightmost chart in Figure 12 shows that all of the horses experienced a significant reduction in spontaneous movement discomfort between the first and second examinations.

[0088] Overall, this study showed a reduction in sensitivity from an average of 2.92 at day 0 to an average of 0.67 at day 90, with a 71% reduction at day 10 and improvement reaching 77% at day 90. On average, joint deformation was 3.77 at day 0 and decreased to 1.78 at day 90. This indicates that it takes a long time for joint deformation to show improvement. At day 10, the improvement in joint deformation was 15%, while at day 90 it improved to 53%. Overall, this study demonstrates that injection of cultured equine gingival fibroblasts according to the present invention has beneficial effects on joint deformation and sensitivity in subjects exhibiting fetlock joint dysfunction, and the animals were well-tolerated.

[0089] Although various illustrative embodiments of the present invention have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the present invention.

Claims

1. A cultured mammalian cell population comprising mammalian gingival fibroblasts, wherein the cells are cultured such that at least about 90% of the cells in the population express CD90 and CD63, and about 20% or less of the cells in the population express CD146.

2. A cultured mammalian cell population according to claim 1, wherein about 10% or less of the cells in the population express at least one mRNA selected from the group consisting of mRNAs of CD146, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IFI44L, RSAD2, IFIT1, IFNA1, and IFNG.

3. The cultured mammalian cell population according to claim 1, wherein at least about 50% of the cells in the population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.

4. The cultured mammalian cell population according to claim 1, wherein the gingival fibroblasts are allogeneic.

5. The cultured mammalian cell population according to claim 1, wherein the gingival fibroblasts are autologous.

6. A pharmaceutical or cosmetic composition comprising a cultured mammalian cell population as described in claim 1, and comprising at least one pharmaceutically or cosmetically acceptable carrier or excipient.

7. A method for culturing a population of mammalian cells, comprising mammalian gingival fibroblasts, wherein at least about 90% of the cells in the population express CD90 and CD63, and about 20% or less of the cells in the population express CD146, and the method is A process for obtaining tissue containing mammalian gingival fibroblasts; A step of digesting the aforementioned tissue with enzymes to create an aggregate of isolated cells; below: (i) A step of culturing the isolated cell aggregate in a culture medium mixture containing at least 20% fetal bovine serum and basic fibroblast growth factor for a first period of time to produce adherent cells; (ii) When the first period has ended, the step of washing the adherent cells and replacing the culture medium mixture; and (iii) A step of culturing the adherent cells over a second period; and (iv) When the second period is completed, the adherent cells are trypsinized to create a subsequent aggregate of isolated cells, and the subsequent aggregate of isolated cells is washed; A process of performing subculturing, including, The process of repeating (i) to (iv) thereby performing multiple passages; and The process of creating a cultured mammalian cell population by, upon completion of the last of the multiple passages, treating the adherent cells with trypsin, washing them, and packaging them for storage. A method of including.

8. The method according to claim 7, wherein the first period is three days or four days.

9. The method according to claim 8, wherein the second period is three or four days.

10. The method according to claim 7, wherein interleukin-1β or a non-essential amino acid is added to the culture medium mixture during one or more of the aforementioned multiple passages.

11. The method according to claim 7, wherein the proliferation of the adherent cells during at least one of the multiple passages exceeds 10 times.

12. The method according to claim 7, wherein the cultured mammalian cell population is cultured so that about 10% or less of the cells in the population express at least one mRNA selected from the group consisting of mRNAs of MCAM, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IFI44L, RSAD2, IFIT1, IFNA1, and IFNG.

13. The method according to claim 7, wherein the cultured mammalian cell population is cultured such that at least about 50% of the cells in the population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.

14. The method according to claim 7, wherein the gingival fibroblasts of the mammal are allogeneic.

15. The method according to claim 7, wherein the cultured mammalian cell population is processed to produce a pharmaceutical product comprising at least one pharmaceutically or cosmetically acceptable carrier or excipient.

16. A method of using a pharmaceutical product comprising or derived from a population of cultured mammalian cells including mammalian gingival fibroblasts, wherein the cells are cultured to treat a disease of a mammal such that at least about 90% of the cells in the population express CD90 and CD63, and about 20% or less of the cells in the population express CD146, the method comprising the step of topically conferring the pharmaceutical product to the mammal or injecting the pharmaceutical product to the mammal.

17. The method according to claim 16, wherein the disease is a skin disease selected from the group consisting of skin wounds, skin aging, alopecia, allergic reactions, atopic dermatitis, asthma, or pressure ulcers, and the step of topically applying the pharmaceutical to the mammal further comprises the step of topically applying the pharmaceutical to the mammal near the skin disease.

18. The method according to claim 16, wherein the disease is an orthopedic condition selected from the group consisting of joint disorders, arthropathy, osteoarthritis, cartilage defects and tendinitis, and the step of injecting the pharmaceutical into the mammal comprises the step of injecting a dose of between 10 million and 30 million cells into the joint or tendon of the mammal.

19. The method according to claim 16, further comprising the step of preparing the pharmaceutical by culturing allogeneic gingival fibroblasts of a mammal.

20. The method according to claim 16, further comprising the step of preparing the pharmaceutical by culturing autologous gingival fibroblasts of a mammal.

21. The method according to claim 16, wherein the disease is a nerve injury, and the step of injecting the drug into the mammal comprises injecting a dose of between 10 million and 30 million cells near the injured nerve.

22. The method according to claim 16, wherein the disease is a tumor, and the step of injecting the pharmaceutical into the mammal comprises the step of injecting a dose of between 10 million and 30 million cells into the tumor.

23. The method according to claim 16, wherein the mammal is selected from the group consisting of humans, horses, dogs, cats, camels, dromedaries, llamas, cattle, goats, and weasels.

24. The method according to claim 1, wherein the gingival fibroblasts of the mammal are selected from the group consisting of humans, horses, dogs, cats, camels, dromedaries, llamas, cattle, goats, and weasels.

25. The method according to claim 7, wherein the gingival fibroblasts of the mammal are selected from the group consisting of humans, horses, dogs, cats, camels, dromedaries, llamas, cattle, goats, and weasels.

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