Mammalian cell populations and agents for mammalian cell therapy and improved cell culture methods

Abstract

The present invention provides a composition of matter comprising a population of cells and an agent, which is derived from human gingival fibroblasts and has a cell phenotype present in a proportion that is not found in the natural gingival tissue, the cell phenotype being preferentially selected to express a protein beneficial to angiogenesis and anti-inflammatory action, meanwhile, cell populations of metalloproteinase formation that promotes tumorigenicity and / or inhibits tissue regeneration are reduced. The present invention provides methods of producing the compositions that can achieve multiple proliferation improvements compared to previously known methods, and methods of using the compositions in a wide range of human cell therapies.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Patent Application Serial No. 18 / 301,864, filed April 17, 2023, U.S. Patent Application Serial No. 18 / 301,856, filed April 17, 2023, and U.S. Patent Application Serial No. 18 / 301,839, filed April 17, 2023. This application also claims priority to U.S. Provisional Patent Application Serial No. 63 / 490,489, filed March 15, 2023, and European Patent Application Serial No. 23305350.3, filed March 15, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention relates generally to compositions of matter derived from gingival fibroblasts and pharmaceutical agents for cell therapy, and improved methods for culturing the compositions to produce populations of specialized phenotypes with advantages over naturally occurring tissues. In particular, the methods of the present invention are designed to enhance cell proliferation to facilitate production of the cell compositions of the present invention in sufficient quantities to enable commercial scale production. The present invention also includes methods of using the new compositions to treat various mammalian diseases, including diseases affecting epithelial and endothelial surfaces (e.g., skin aging, pressure sores, hair loss, immune and vascular diseases) and diseases of osteoarthritic origin (e.g., osteoarthritis, cartilage defects, etc.). BACKGROUND

[0004] Gingival fibroblasts have been investigated for a wide range of cell therapies for at least the past two decades due to their multipotent nature and the ability of the cells to differentiate into a more extensive spectrum of cell types compared to other types of mesenchymal stem cells. Moreover, while the latter are collected from bone marrow, this poses some risk to the donor, gingival fibroblasts are readily collected from gingival tissue, such as during wisdom tooth extraction and other dental procedures.

[0005] The use of gingival fibroblast (GF)-derived compositions (cultured GF cells and GF-conditioned medium) for the treatment of a number of mammalian ailments has been described, as set forth in the following U.S. patents and publications: U.S. Patent No. 8,609,085 (atherosclerosis); U.S. Patent No. 8,303,948 (skin wounds); U.S. Patent Application Publication No. 2011 / 0097421 (skin aging); U.S. Patent No. 10,624,838 (hair loss); U.S. Patent No. 11,229,670 (allergic reactions, atopic dermatitis, and asthma); U.S. Patent Application Publication No. 2018 / 0028571 (cancer); U.S. Patent Application Publication No. 2016 / 0256496 (human orthopedic pathologies, including osteoarthritis and cartilage defects); U.S. Patent Application Publication No. 2020 / 0268805 (equine musculoskeletal diseases, including tendonitis, arthrosis, and joint degeneration), and International Publication No. WO 2023 / 007244 Al (pressure sores). All of the following patents and applications are assigned or licensed to the assignee of the present application, the entire contents of which are incorporated herein by reference.

[0006] Methods of culturing gingival fibroblasts are described in a number of previously known articles, 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); Ferré 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 foregoing articles describe previously known methods of culturing cells, including the use of at least one culture medium, such as Eagle's minimally essential medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM), antibiotics (such as penicillin, streptomycin, or gentamicin), and serum derived from mammalian blood sources, such as 10% fetal bovine serum (FBS).

[0007] Generally, previously known methods of culturing gingival fibroblasts include removal of gingival tissue, followed by mincing or enzymatic digestion. The cells are then incubated in a humidified incubator with a 5% carbon dioxide atmosphere for a period of about two to three weeks until the cells reach confluence, after which they are trypsinized to produce a single cell suspension. In some culturing methods, the culture medium can be refreshed every 72 hours, and after the initial culturing period, the gingival fibroblast colonies can be again trypsinized to produce a single cell suspension, which is seeded into fresh culture to continue incubation for a period of two weeks, a process commonly referred to as "passaging." After the final passaging and trypsinization step, the cultured cells can be washed and collected for the desired application scenario.

[0008] There are a number of disadvantages to previously known culturing methods and resulting cell compositions. One such disadvantage is the length of time required to culture a small number of gingival fibroblasts to form a large scale cell colony sufficient to support commercial applications. It would be particularly desirable to provide a cell composition that enhances cell proliferation during the culturing phase and a method for culturing the same. In this way, a cell population sufficient for commercial methods of treating the disease conditions described in the above-incorporated patents can be achieved, thereby facilitating the transition of the use of gingival fibroblasts from research laboratory techniques to commercial scale practical therapeutic applications.

[0009] Another disadvantage of previously known compositions and methods is the inability to tailor the protein expression profile of the cell phenotype in the cultured cell population. For example, it is known that native gingival fibroblasts will contain a large population of cells that express CD146 (also known as melanoma cell adhesion molecule, which is associated with the tumorigenicity of melanoma cells) when cultured in vitro. Thus, for many of the treatments described in the above-incorporated patents and applications, it is desirable to limit the expression of the CD146 phenotype. Similarly, it is also desirable to culture gingival fibroblasts to enhance the expression of certain phenotypes, such as CD90 (also known as THY1) and TIMP1, the latter of which inhibits the production of matrix metalloproteinases and reduces the inflammatory response.

[0010] In view of the foregoing, there is a need to provide compositions and medicaments derived from gingival fibroblasts that include cell populations cultured to have a specific protein expression phenotype, wherein the phenotype is preferentially selected to express proteins that favor angiogenesis and anti-inflammatory effects, while reducing cell populations that form metalloproteinases that promote tumorigenicity and / or inhibit tissue regeneration.

[0011] There is also a need to provide sufficient amounts of the compositions, medicaments, and methods for culturing the compositions to facilitate methods of using the compositions in the treatment of a variety of mammalian diseases, including but not limited to atherosclerosis, skin wounds, skin aging, hair loss, allergic reactions, atopic dermatitis, asthma, pressure sores, cancer, orthopedic lesions including osteoarthritis and cartilage defects, tendonitis, arthrosis, and joint degeneration. SUMMARY

[0012] The present invention relates to compositions, medicaments, and methods of producing compositions of gingival fibroblasts having a phenotype cultured to express preferred proteins. In particular, unlike gingival fibroblasts present in native gingival tissue, the cell populations of gingival fibroblasts of the present invention are preferentially selected to express a cell phenotype that expresses proteins that favor angiogenesis and anti-inflammatory effects, while reducing cell populations that form metalloproteinases that promote tumorigenicity and / or inhibit tissue regeneration. In one embodiment, unlike gingival fibroblast populations present in native gingival tissue, the compositions have a cell population or conditioned media in which at least about 90% of the cells have an expression phenotype of THY1, CD99, or CD63, and about 20% or less of the cells have an expression phenotype of CD146.

[0013] According to another aspect of the present invention, methods for culturing the compositions having the above-described specific phenotypic characteristics that enhance cell proliferation to actually produce commercial scale amounts of the compositions are provided. In particular, the methods of the present invention not only advantageously preferentially select a cell phenotype, but also allow culturing at significantly increased cell proliferation rates. In one preferred embodiment, the cell culturing process includes multiple passages of the cells in 20% FBS with non-essential amino acids and frequent media changes, resulting in cell proliferation rates that are more than doubled over prior art culturing methods.

[0014] Methods of using the compositions and medicaments of the present application to treat a range of mammalian diseases including atherosclerosis, skin wounds, skin aging, hair loss, allergic reactions, atopic dermatitis, asthma, cancer, human orthopedic lesions including osteoarthritis and cartilage defects, tendonitis, joint disease and joint degeneration, and pressure sores are also provided in accordance with the present application. The cultured gingival fibroblasts and conditioned media of the present application can also be used to regenerate excised or damaged nerves, such as spinal cord injuries. Based on preliminary animal data, it has been observed that the response to the foregoing treatments can be dose dependent, such that to achieve a clinically significant and lasting therapeutic response, the treatment should deliver a population of between 50 million and 400 million cells to the treatment site, and more preferably about 200 million cells.

[0015] The compositions of the present application can comprise a population of cultured mammalian cells as defined above in a liquid cell culture medium, or a medicament derived from a population of cultured mammalian cells as defined above, or a pharmaceutical or cosmetic composition comprising a population of cultured mammalian cells as defined above, said compositions comprising at least one pharmaceutically or cosmetically acceptable carrier or excipient.

[0016] Other features of the present systems and methods will be apparent from consideration of the following description and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Table 1 is a table showing the evolution of the gingival fibroblast phenotype in the population of cultured cells during five culture passages according to the principles of the present application.

[0018] Figure 2 Table 2 is a table showing the representative protein expression profiles and proliferation indices of various conventional media compared to the media of the present application.

[0019] Figure 3 Table 3 is a graph showing the effect of using different concentrations of FBS during cell culture, and changing the media during the first passage of cell culture.

[0020] Figure 4 Table 4 is a graph showing the effect of changing the media during the second passage of cell culture.

[0021] Figure 5 Table 5 is a graph showing the effect of culturing gingival fibroblasts with and without non-essential amino acids on the phenotype of the cell population during additional culture passages.

[0022] Figure 6 Table 6 is a series of bar graphs showing the data obtained from the treatment day and one year follow-up period in a canine model using different doses of the compositions of the present application.

[0023] Figure 7 is a series of bar graphs showing the percent efficacy of treatments performed as indicated in Figure 6

[0024] Figure 8 is a series of bar graphs showing the dose dependency and persistence of treatments performed as indicated in Figure 6

[0025] Figure 9 is a table showing the summary of data obtained at ninety days after injection of a composition according to the application to treat fetlock joint disease in a first group of horses.

[0026] Figure 10 is a table showing the summary of sensitivity data obtained at ninety days after injection of a composition according to the application to treat tendinopathy in a second group of horses.

[0027] Figure 11 is a table showing the summary of joint deformation data observed at ninety days after injection of a composition according to the application to treat tendinopathy in a second group of horses.

[0028] Figure 12 is a series of bar graphs showing the improvement in health scores in a second group of horses as a function of the number of days from the date of treatment. DETAILED DESCRIPTION

[0029] The present invention relates to compositions of matter derived from gingival fibroblasts having phenotypes present in ratios not found in native gingival tissue, including cell populations and pharmaceutical agents. The invention also provides methods of producing the compositions, and methods of using the compositions and conditioned media in a wide range of mammalian, including human, canine, equine, and feline, cell therapies.

[0030] Compositions and medicaments of the invention

[0031] Compositions according to the principles of the invention preferably include cell populations expressing angiogenic and anti-inflammatory proteins, with few cells having phenotypes associated with tumorigenicity or promoting metalloproteinase formation. As described below, cell populations are cultured from gingival fibroblasts to have phenotypic ratios different from those found in native gingival tissue. Cell populations of the invention can be packaged directly for use as pharmaceutical agents, or used to produce conditioned media for use as pharmaceutical agents.

[0032] ​​In a preferred embodiment, the composition comprises about 10% or less of a cultured mammalian cell population expressing at least one mRNA selected from the group consisting 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 mRNA.

[0033] Preferably, at least about 50% of the cultured mammalian cell population expresses at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNA; more preferably, at least about 90% of the cultured mammalian cell population expresses CD63 mRNA and TIMP1 mRNA. TIMP1 represents TIMP metallopeptidase inhibitor 1.

[0034] The applicant observed in laboratory tests that the protein expression characteristics of CD63 and TIMP1 in cultured gingival fibroblasts according to the invention (measured by mRNA transcripts) differed significantly from those of these proteins produced by natural gingival tissue. As described below, the method of the invention for culturing gingival fibroblasts in several culture passages preferentially enhances the desired phenotype while limiting the growth of less desirable phenotypes.

[0035] Figure 1 This is a graph showing the evolution of specific cellular protein expression phenotypes during multiple passages of human gingival fibroblasts to produce cell populations according to the invention. Specifically, the phenotypic proportions of naturally occurring gingival tissue comprise a population in which only 30.2% of cells express the desired CD90 protein, and 6.9% express the desired CD63, while 20.7% express the less desirable CD146 protein (a melanoma cell adhesion molecule). However, as shown in subsequent passages 0 through 5, the proportion of the desired phenotype preferentially increases, while the proliferation of the undesirable phenotype is limited. For example, at the completion of the second passage, the proportion of cells expressing CD90, CD105, CD73, and CD63 proteins all exceed 95%, while the proportion of cells expressing CD146 is 13.4%. However, during the next two passages, the proportion of cells expressing CD146 decreases by more than half (to 6.2%), while the proportion of cells expressing CD90, CD105, CD73, and CD63 proteins does not decrease significantly.

[0036] Preferably, substantially all cells in the cultured mammalian cell population have an undifferentiated phenotype, more preferably a fibroblast-like phenotype. The cultured mammalian cell population is preferably derived from mucosa, more preferably from oral mucosa, particularly from the buccal mucosa, lingual mucosa, palatal mucosa, lip mucosa, sublingual mucosa, or gingival mucosa. Procedures for obtaining samples or performing biopsies from mucosa are well known to those skilled in the art. Generally, biopsy cells are separated by enzymatic digestion prior to amplification.

[0037] Suitable cultured mammalian cell populations can be derived from any mammal (e.g., livestock), such as those selected from the group consisting of: equines (especially horses), camels (especially camels, dromedaries, or llamas), bovids, capillaries, canids (especially dogs), felines (especially cats), and mustelids. For compositions intended for human use, cultured mammalian cell populations are preferably obtained from human cell populations. The cell populations according to the invention can be autologous or allogeneic. As those skilled in the art will understand, the individuals from which the cells are obtained and the individuals to whom the cells are administered are preferably of the same species; however, they can also be different species.

[0038] Cell populations can exist in various forms, such as suspensions in liquids (e.g., physiological solutions, PBS, or cell culture media), cultures on solid media, or precipitates (especially centrifuged precipitates).

[0039] As used herein, compositions or agents according to the invention comprise a cell population according to the invention in a preventive or therapeutically effective amount. Preferably, the compositions according to the invention comprise 5 million to 40 million cells according to the invention, more preferably 10 million to 30 million cells according to the invention, even more preferably 15 million to 25 million cells according to the invention, and most preferably about 20 million cells according to the invention. The quantification of cells according to the invention can be performed by any method known to those skilled in the art for counting mammalian cells, such as a cell counter.

[0040] The composition or agent can be in any form suitable for its intended application. In a preferred embodiment of the invention, the composition can be in an injectable form, for example, suitable for delivery by injection with a syringe. In an alternative embodiment, the composition can be in a form suitable for topical application. In this case, the composition is preferably in the form of a lotion, cream, ointment, gel, spray, wipe, dressing, pad, or patch.

[0041] As used herein, the "conditioned medium" product is derived from the mammalian cell population of the present invention and may be any product obtained from the cell population itself or any product containing secretions from the cell population. Preferably, the conditioned medium product derived from the cultured mammalian cell population is a cell extract. The extract can be obtained by any cell fragmentation method known in the art, including those derived from the group consisting of membrane extracts, cytoplasmic extracts, or nuclear extracts.

[0042] The conditioned medium according to the invention may further comprise a liquid cell culture medium that has been contacted with the cells of the cell population of the invention, particularly for a period of time sufficient to allow the cells to secrete in the culture medium. Thus, the conditioned medium according to the invention contains cell secretions from a mammalian cell population cultured according to the invention. Preferably, the cells are contacted with the culture medium for at least 2, 4, 6, 8, 10, 12, 24, 36, or 48 hours, and typically less than 56 or 72 hours.

[0043] The conditioned medium according to the invention can undergo processing steps, such as centrifugation, filtration, or concentration. In particular, the conditioned medium according to the invention can be a concentrated conditioned medium, and more particularly a conditioned medium concentrated 2, 5, 10, 25, or 50 times relative to its source unconcentrated conditioned medium.

[0044] This document describes cells, cell populations, phenotypes, etc., that express or do not express a specified protein or mRNA. According to this disclosure, a specified cell is considered not to express the specified protein or mRNA when standard laboratory assays (e.g., single-nuclear RNA sequencing methods (snRNAseq), such as those described essentially by Grindberg et al., Proceedings of the National Academy of Sciences (Proc. Natl Acad. Sci. USA) 110:19802-19807 (2013), fluorescence-activated cell sorting (FACS), etc.) fail to detect a perceptible level of the protein or mRNA. Conversely, when the methods detect a perceptible level of the protein or mRNA, the cell is considered to express the specified protein or mRNA.

[0045] As those skilled in the art will understand, protein expression can be measured by measuring the corresponding mRNA transcripts according to this disclosure.

[0046] Culture of preferred cell populations

[0047] According to one aspect of the invention, gingival fibroblasts, after being extracted from natural gingival tissue, are passaged five to seven times to preferentially modulate the phenotype in the composition, thereby increasing desired protein expression characteristics while suppressing undesired protein expression characteristics.

[0048] In one embodiment, xenologous mammalian gingival tissue is collected from the oral cavity of a suitable mammalian species (i.e., the species from which the cultured cell composition or conditioned medium will ultimately be used). The collected tissue is cut into small fragments using a scalpel, and the fragments are enzymatically digested. The dissociated cells are washed in phosphate-buffered saline (PBS), and a cell suspension is generated by placing the washed cells in a mixture of culture medium and 20% FBS. For example, suitable culture media according to the invention include Eagle's minimum essential medium (MEM or EMEM), Eagle's minimum essential medium α-modified (αMEM), and Eagle's basal medium (BME).

[0049] Next, at 10,000 cells / cm 2 Cells were cultured at zero seeding density in culture flasks containing culture medium, 20% FBS, antibiotics, and basic fibroblast growth factor (bFGF). Basic fibroblast growth factor (bFGF) refers to human bFGF, more preferably recombinant human bFGF produced in *E. coli*, having the sequence identified in SEQ No. 1, the sequence of which is listed in U.S. Patent No. 7,951,593, which is incorporated herein by reference. The culture medium was removed, and the cells were washed with PBS on days 3 and 10. On day 21, the culture medium was removed, the cells were washed again with PBS, and they were trypsinized to remove them from the flasks. At the end of zero passage (after 21 days), the gingival fibroblast population typically expanded to 6-fold.

[0050] In the first passage, cells were cultured in a mixture of medium, 20% FBS, and bFGF at 4,500 cells / cm² in culture flasks under an atmosphere of 37°C, 95% oxygen, 5% carbon dioxide, and 95% humidity. 2 Cells are cultured at a seeding density of [number missing]. On day 3, cells are washed with PBS, provided with a fresh mixture of culture medium, 20% FBS, and bFGF, and cultured for another 3 days, after which the cells are triedinized again. During the first passage, which lasts 6 days, the cell population typically expands approximately 15-fold, for example, from approximately 10 million cells to approximately 143 million cells in the exemplary embodiment. After further washing with PBS, the cells are placed in a storage medium consisting of a solution of approximately 4% w / v human serum albumin (USP / Ph.Eur) and 10% DMSO (USP / Ph.Eur), and can be stored in quantities of 10 million cells per storage tube.

[0051] In the second passage, cells from a single storage tube were introduced at a rate of 4,500 cells / cm³. 2Cells were seeded at a density dispersed in a mixture of culture medium, 20% FBS, and bFGF, which was refreshed after 4 days. On day 7, the culture medium mixture was removed. By the time the second passage was completed, the cells had typically expanded approximately 15-fold again.

[0052] In the third passage, cells were placed in a mixture of culture medium, 20% FBS, and bFGF at a density of 4,500 cells / cm². 2 The cells are seeded at a density in, for example, a multi-plate bioreactor or other similar apparatus for large-scale expansion. After 3 days, the culture medium mixture is replaced with a fresh mixture, and the cells are cultured for another 3 days. After 6 days, the cells are trypsinized, washed in PBS, and placed in storage tubes containing a storage medium consisting of approximately 4% w / v human serum albumin (USP / Ph.Eur) and 10% DMSO (USP / Ph.Eur). By the completion of the third passage, the cells will typically have expanded to approximately 15-fold. Preferably, the cells can be dispensed for storage in tubes containing approximately 10 million cells each.

[0053] In the fourth passage, cells from a single storage tube were introduced at a rate of 4,500 cells / cm³. 2 Cells were seeded at a density dispersed in a mixture of culture medium, 20% FBS, and bFGF. This mixture was refreshed after 4 days, washed with PBS, and placed in a bioreactor according to the second passage protocol for a fifth passage over an additional 6 days. At the end of the fourth passage, cells typically expanded approximately 15-fold and were trypsinized, washed with PBS, and prepared for further culture in the fifth passage.

[0054] In the fifth passage, cells were placed in a mixture of culture medium, 20% FBS, and bFGF at a density of 4,500 cells / cm². 2 The cells are inoculated at a density in a bioreactor or other large-scale amplification device, and the mixture is replaced with a fresh culture medium mixture after 3 days. On day 3, the removed culture medium mixture is replaced with a fresh culture medium mixture omitting fetal bovine serum and adding interleukin-1β (IL-1β). One day later, the culture medium mixture is removed, and the cells are typically amplified approximately 15-fold and trypsinized, washed with PBS, and stored in storage tubes containing 10 million cells each, each containing the storage medium as described above.

[0055] Preferably, as described above, the cell population of the present invention needs to be cultured in the presence of inflammatory cytokines (such as IL-1β) during the fifth passage culture before it can be used for the therapeutic or cosmetic applications of the present invention. The concentration of inflammatory cytokines, particularly IL-1β, in the culture medium can be from 0.1 ng / ml to 10 ng / ml, more preferably from 0.5 ng / ml to 2 ng / ml. Before use in the therapeutic or cosmetic applications according to the present invention, inflammatory cytokines, particularly IL-1β, can be recovered from the cell culture, for example, by washing the cells.

[0056] The cell cultures of the present invention can be concentrated, for example, by filtration or by centrifugation. In particular, the cell cultures can be concentrated 2, 5, 10, 25 or 50 times relative to the unconcentrated cell cultures from which they originated.

[0057] The composition according to the present invention is produced by cultivating it using the above method. Figure 2 An exemplary cell population with the protein expression characteristics described herein. While not intended to be limiting, key differences in the process of culturing gingival fibroblasts according to the present invention compared to the culture methods described in previously known methods include the use of 20% FBS and basic fibroblast growth factor during cell culture, and the replacement of the culture medium mixture approximately every predetermined time during multiple passages lasting six to seven days. Furthermore, the method also 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, such as… Figure 1 As shown.

[0058] Now for reference Figure 2 Another key aspect of this invention is the ability of the above-described method to enhance cell proliferation during the culture process. This index, also known as the amplification index, indicates how many times the cells have expanded during a specified passage period. It has been observed that the method of this invention achieves significantly greater proliferation compared to the cell culture methods described in the aforementioned literature. This, in turn, allows the compositions of this invention to be produced in quantities sufficient to support commercial applications within a specified time period. Specifically, Figure 2 Results obtained after five passages using standard medium containing 10% FBS and Scarcell medium are reported. Exemplary formulations for the latter, as described above, include DMEM, 20% FBS, and bFGF. While not intended to limit the methods of action, it is assumed that higher concentrations of fetal bovine serum (FBS) and frequent FBS replacement are used to maintain cell culture stability.

[0059] For conventional culture media, it consists of various combinations of DMEM containing and without non-essential amino acids, Lysat 3%, heparin, and 10% FBS. Figure 2The proliferation index varied from approximately 5 to 9 times. In contrast, when using Scarcell medium, the proliferation index of gingival fibroblasts from one source reached 17 times, and from another source it was as high as 22 times. Furthermore, as described above, the method according to the invention increased the yield of cells desired to express the CD63 protein by approximately 20 to 50%. These results demonstrate a two- to three-fold increase in the ability to expand gingival fibroblasts during culture, thereby providing a pathway to supply more of the composition of the invention over a specific time period and potentially reducing the cost of such production.

[0060] Now for reference Figure 3 and 4 This diagram depicts how the increase in the gingival fibroblast population changes over time and in the culture medium. Specifically, Figure 3 The lines represented by hollow squares (□) and triangles (▲) show the evolution of cell numbers cultured in medium with 10% FBS during the first passage. The lines represented by triangles (▲) correspond to cultures where the medium was changed after the first 4 days, while the lines represented by hollow squares (□) correspond to cultures where the medium was not changed. Figure 3 In the figure, the lines represented by rhombuses (◆) and solid squares (■) show the evolution of cell mass during the first passage in a medium with 20% FBS, where the medium was changed to solid squares (■) after the first four days, or the medium was not changed to rhombuses (◆). Figure 3 All four lines show that the number of viable cells plateaued or decreased after 7 days of culture, with 10% FBS medium (with medium change) providing approximately the same cell count as that achieved using 20% ​​FBS (without medium change). However, unexpectedly, using 20% ​​FBS while also changing the medium after the first 4 days resulted in a doubling of the number of viable cells after 7 days of culture.

[0061] Figure 4 Provided similar Figure 3 The results are shown, but a second culture was performed in medium with 20% FBS. Specifically, in Figure 4, the line represented by the diamond (◆) corresponds to medium with 20% FBS, where the medium was not changed after 4 days, and the line represented by the solid square (■) shows the evolution of cell number cultured in medium with 20% FBS, where the medium was changed after the first four days. Figure 4 As shown, the number of viable cells without changing the culture medium began to decline after 6 days and began to plateau after 7 days with changing the culture medium. Notably, and unexpectedly, changing the culture medium 4 days into the second culture passage period approximately doubled the viable cell yield during the second passage period.

[0062] Turn nowFigure 5 Alternative embodiments of the method according to the invention are described, wherein one or more additional culture passages are performed to reduce the cell population expressing the unwanted protein CD146. In this method, cells that have undergone a fourth passage as described above are further cultured for a fifth, sixth, and optionally a seventh passage, the procedure being essentially as described above, except that non-essential amino acids are added to the culture medium. Figure 5 As shown, when cultured with NEAA, the percentage of cells expressing CD146 protein increased from 27.9% at the end of the fourth passage to 52.7% in the fifth passage, before decreasing to 14.2% in the sixth passage. In contrast, omitting NEAA from the culture medium during the fifth passage increased the percentage of cells expressing CD146 to 74.4%, which decreased to only 25.8% after two more passages. Therefore, the method of the present invention preferably includes adding NEAA to the culture medium during late passages of the cell culture process to specifically adjust the proportions of the cell population to alter the protein expression profile.

[0063] Therapeutic uses of compositions of the invention

[0064] Cell cultures and conditioned media prepared according to the principles of the present invention can be advantageously used to treat a variety of mammalian diseases, as described in the aforementioned incorporated common assignments or licenses and publications. For example, cultured cell suspensions or conditioned media products derived therefrom can be used to treat atherosclerosis, skin wounds, skin aging, hair loss, allergic reactions, atopic dermatitis, asthma, cancer, human orthopedic diseases (including osteoarthritis and cartilage defects), tendinitis, arthropathy and joint degeneration, and pressure sores. The compositions and conditioned media of the present invention can also be used for the regeneration of nerve tissue, for example, to treat spinal cord injuries. Administration of the cultured mammalian cell population as defined above, or conditioned media products derived from the cultured mammalian cell population as defined above, or pharmaceutical compositions derived therefrom, can be carried out by any method known in the art, including subcutaneous, intravenous, intramuscular, intradermal, or topical application, near or on the skin area to be treated.

[0065] For example, skin-related conditions can be treated by topical application of the composition or conditioned medium, while vascular diseases can be treated by intravascular injection or delivery of a stent or other device with a reservoir capable of delivering the composition. Other applications (such as bone disease treatment) may involve direct injection near joints or tendons.

[0066] A surgical wound is an artificially created wound during the course of surgery. Such surgical wounds include, in particular, wounds or scar repair wounds (such as hypertrophic scars) that occur during plastic and reconstructive surgeries. Plastic and reconstructive procedures can be of any type, such as breast surgery, abdominal surgery, nasal surgery, ear surgery, or the removal of skin defects. As anticipated in this article, skin defects involve abnormal skin formation found in genetically susceptible individuals or are the result of abnormal skin development during embryogenesis, and particularly include giant nevi, cleft lip, and keloids.

[0067] As described in this article, “treating skin wounds” involves promoting, accelerating, or improving the healing of the injured site, that is, forming functional skin at the site of injury. As anticipated in this article, “functional skin” refers to skin that has recovered its mechanical properties and barrier function relative to uninjured areas. Inflammatory skin lesions are typically caused by inflammatory skin diseases, particularly chronic inflammatory skin diseases, and can include groups consisting of dermatitis, inflammatory rashes, ichthyosis, and psoriasis. Chronic inflammatory diseases also include rheumatoid arthritis, lupus erythematosus, and multiple sclerosis.

[0068] Cultured cell suspensions or conditioned medium products derived therefrom can also be used for cosmetic purposes. As contemplated herein, "cosmetic-acceptable carriers or excipients" refers to any material suitable for cosmetic compositions. Preferably, cosmetic-acceptable carriers or excipients are suitable for topical application. Cosmetic-acceptable carriers or excipients according to the invention include, but are not limited to, any 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.

[0069] Example 1

[0070] Single-nuclei RNA sequencing of cell populations of the invention

[0071] The cell population of the present invention generated according to the above method was subjected to single-nuclear RNA sequencing (snRNAseq). The snRNAseq method was originally described in Greendberg et al., Proceedings of the National Academy of Sciences of the United States of America 110:19802-19807 (2013) and is known to those skilled in the art. The results of the analysis are presented in the table below.

[0072]

[0073]

[0074]

[0075] Example 2

[0076] Proteins expressed by cell populations of the invention

[0077] The expression of certain proteins was determined by fluorescence activated cell sorting (FACS), as illustrated in the table below.

[0078] Proteins Percentage of cells expressing a protein CD34 <1 CD45 6 CD63 97 CD90 97 CD99 97

[0079] Results of animal studies

[0080] Initial testing of the compositions produced according to the method of the invention has been conducted in canine and equine models, specifically for the treatment of osteoarthritis in a group of 95 dogs, and for the treatment of individual groups of horses suffering from arthropod arthritis and tendinopathy. Regarding Figures 6 to 12 A summary of the results obtained during those tests was discussed.

[0081] Treatment of secondary osteoarthritis in dogs

[0082] Osteoarthritis is a common and well-known disease in canine veterinary medicine. This type of disease is usually caused by damage to the subchondral soft tissue, articular cartilage, or a combination of both. This triggering leads to degeneration of the articular cartilage, which involves changes in bone and tissue. The specific origin of the lesion (such as inflammation of the joint or synovial lining, or a disease that causes joint degeneration) does not determine the final outcome. Instead, synovitis promotes the production of pro-inflammatory cytokines and metalloproteinases, thus driving the chronic progression of the disease. Furthermore, synovitis causes joint pain and synovial effusion, leading to joint instability. The prevalence in dogs within one year ranges from 2% to 20%. Osteoarthritis causes significant comorbidities, is often associated with obesity, and requires long-term treatment with anti-inflammatory medications or joint replacement surgery.

[0083] Current treatments for osteoarthritis in canines do not actively promote cartilage regeneration and primarily focus on symptom relief, such as reducing pain caused by joint inflammation. Recent research into alternative therapies has advanced the development of cell-based therapies, mainly due to their potential regenerative potential. Current allogeneic cell therapies are limited by their half-life after administration to the individual. The aim of this study was to evaluate the safety and efficacy of a cell therapy based on allogeneic gingival fibroblast cultures used in domestic dogs with osteoarthritis.

[0084] A randomized study of 95 dogs was conducted using a single intra-articular injection to investigate the efficacy, safety, and tolerability of canine gingival fibroblasts in the treatment of osteoarthritis. The injection medium consisted of 10% DMSO, 88% DMEM, 1% L-glutamine, and 1% non-essential amino acids, with a total volume of 1 mL. Sixteen dogs received only the injection medium without gingival fibroblasts (placebo), 28 dogs received an injection of 2 million cultured cells, 28 dogs received an injection of 5 million cultured cells, and 23 dogs received an injection of 10 million cultured cells. Study parameters were as follows: Inclusion criteria were dogs with moderate to severe mono- or poly-osteoarthritis that were refractory to standard treatment; exclusion criteria included sarcoma, a history of prior cell therapy, surgery, or multiple previous injections.

[0085] The dog should be evaluated by a veterinarian one month after the injection and by the pet owner twelve months after the injection. Lameness and pain should be scored according to the following criteria:

[0086]

[0087] More specifically, clinical efficacy was assessed using the Canine Brief Pain Inventory (CBPI) questionnaire, as described by 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), which included pain intensity scores (CBPI questions 1-4), pain interference scores (CBPI questions 5-10), and a total CBPI score. Clinical efficacy was assessed at 1 month and 12 months. Intermediate time points (three months, six months, and nine months) were determined using the Kaplan-Meier methodology (dogs with observed efficacy for up to six months were counted at the three-month and six-month time points).

[0088] Research findings combined Figures 6-8 Discussion was held. One month after the injection, limping improved in all groups. Figure 6 However, no improvement was observed in the placebo group (only a 23% reduction), while the limping was reduced by 52%, 53%, and 41% in the 2 million cell group, 5 million cell group, and 10 million cell group, respectively. Figure 7 All doses showed significant benefits over placebo at one month.

[0089] One month after injection, pain improved by 37%, 49%, 41%, and 45% in the placebo group, the 2 million cell group, the 5 million cell group, and the 10 million cell group, respectively. Figure 6 and 7 No statistically significant differences in pain were observed among the 2 million, 5 million, and 10 million cell groups. The placebo group showed a transient effect, possibly due to the reduction of inflammation caused by flushing the joint with saline solution prior to injection to remove debris. This effect was less than that obtained from injecting gingival fibroblasts.

[0090] No improvement was observed with any treatment for any other clinical parameters (i.e., muscle condition, joint effusion, and joint crepitus) observed before and one month after injection. Figure 6 and 7 However, for all parameters, all doses used in the cell therapy showed significant effects before and after treatment; animal sex did not affect the efficacy of the treatment. No adverse events were observed during the first 72 hours. At one month, efficacy in all groups was not affected by the dog's weight, age, or initial degree of lameness, and no correlation was observed between the dog's initial clinical condition and its age or weight prior to injection.

[0091] One year post-injection, clinical efficacy was assessed based on the CBPI questionnaire (completed by dog ​​owners) 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 cell, 5 million cell, and 10 million cell groups, respectively. All cultured cell groups showed significant differences from the placebo group, with the 10 million cell group demonstrating a significantly longer efficacy than the 2 million and 5 million cell groups. Figure 8 As shown, the percentage of dogs observing efficacy 12 months after injection was 12.5%, 25%, 40%, and 70% for the placebo group, the 2 million cell group, the 5 million cell group, and the 10 million cell group, respectively. All cell groups showed significant differences at 12 months compared to the placebo group, and the 10 million cell group showed a significant difference compared to the 2 million cell group and the 5 million cell group at 12 months.

[0092] Arthroscopic examination was performed on a dog before and four months after injection. The dog had a non-smooth cartilage margin with missing joint ultrastructure (i.e., no clear gaps and a mixture of inflammatory cells and chondrocytes). Four months after injection, arthroscopic histology showed smooth cartilage with normal cartilage ultrastructure (i.e., chondrocytes located in well-defined cavities). This case confirms the clinical observation of cartilage repair achieved after intra-articular injection of cultured cells prepared according to the present invention.

[0093] Dosage-response studies in canines showed that intra-articular injections of 2 million, 5 million, and 10 million cells were more effective than placebo in treating dogs with osteoarthritis at one and 12 months post-treatment. While all three doses showed similar improvement in lameness at one month, at 12 months, the 5 million and 10 million cell doses showed statistically significant differences compared to placebo, while the 2 million cell dose showed no difference. The observed success rate of over 50% within one year is presumably related to cartilage repair.

[0094] Individual variability in studies conducted in uncontrolled environments is similar to that in human populations, thus demonstrating the transferability of preclinical findings to humans. Notably, the studies were conducted in a cohort of dogs that differed significantly in breed, genetic background, sex, and weight / size. Clinical efficacy lasting up to one year in a non-canine population demonstrates the significant potential and robustness of the compositions of this invention in the treatment of knee osteoarthritis.

[0095] Treatment of tendinopathy in equids

[0096] The aim of this study was to evaluate the tolerability and efficacy of local injection of gingival fibroblasts in treating acute tendinopathy of the deep flexor tendons in ten sporting horses (including mares and steeds, racehorses and eventing horses). The study involved a single intratendonal injection of 20 million cultured equine gingival fibroblasts in suitable culture medium at a dose of 2.5 mL. Clinical examinations were performed before injection and 90 days later. Clinical assessments were scored considering deformity, sensitivity, and fever (each graded from 1 to 3, with 3 being the most severe), as well as dynamic lameness assessment (graded from 1 to 5, with 5 being the most severe). Ultrasound examination of the affected limb was also performed to identify hypoechoic areas, where 5 indicates higher hypoechoicity, representing severe inflammation.

[0097] A single injection administered via syringe consisted of: 2.5 mL of 1% sodium hyaluronate solution, 25 mg of sodium chloride, disodium phosphate, sodium dihydrogen phosphate, water, 20 million cultured equine gingival fibroblasts, 200 μL of pure conditioned medium, and 24-hour paracrine secretions from the gingival fibroblasts. The 500 mL pure conditioned medium consisted of L-glutamine, 2400 mg / L sodium bicarbonate, HEPES buffer, sodium pyruvate, hypoxanthine, thymidine, trace elements, growth factors, and 1.1 mg / L phenol red. The heterologous gingival fibroblasts in the suspension were obtained from gingival biopsies of newborn foals provided by volunteer owners.

[0098] For detailed research results, please refer to [link / reference]. Figure 9 It includes detailed illustrations of the scoring system. Specifically, such as... Figure 9 As shown, at three months, ultrasound evolution revealed complete disappearance of hypoechoic lesions in five horses (EC1_2, EC1_4, EC1_5, EC1_8, and EC1_10), areas initially observed by ultrasound prior to injection. Four horses (EC1_1, EC1_3, EC1_7, and EC1_9) showed persistent presence of some hypoechoic lesions; one horse (EC1_6) showed significant persistence of a hypoechoic lesion. Favorable ultrasound evolution of tendon lesions observed three months post-injection persisted at the six-month follow-up.

[0099] Ultrasound and clinical evolution were assessed more than 6 months later: six horses experienced recurrence of tendinitis between 7 and 11 months post-injection; two horses were disqualified for other pathological reasons; and one horse (evening horse, EC1-7) returned to normal activity. All controlled galloping horses experienced recurrence of tendinitis before 12 months. The racehorse (EC1-8) showed favorable evolution of its tendinitis at 12 months and participated in eight races after recovery, achieving three wins and four second-place finishes. Furthermore, although the deformities noted before injection persisted at the 90-day assessment, ultrasound examination revealed significant lesion filling, all horses were lame, and very few exhibited fever or tenderness.

[0100] This experiment demonstrates that the gingival fibroblast composition cultured according to the principles of this invention has a good ability to resolve equine tendinopathy.

[0101] Treatment of carpometacarpal joint disease in equids

[0102] The aim of this study was to evaluate the tolerability and efficacy of local injection of equine gingival fibroblasts cultured according to the principles of this invention in the treatment of ball-and-socket joint disease in racehorses. Ten horses participated in the trial. The ball-and-socket joint refers to the joint where the spur, proximal sesamoid bone, and first phalanx meet.

[0103] This study used a single intra-articular injection of 20 million cultured gingival cells in a suitable culture medium, with examinations performed on days 3, 10, and 90. The injection of 20 million cultured cells was prepared as described above for tendinopathy research. Inclusion criteria were clinical assessment of passive flexion, lameness, joint deformity, and tenderness. Passive flexion, lameness, joint deformity, and sensitivity were examined on days 3, 10, and 90. Favorable outcomes included resumption of activity, resumption of training, and resumption of competition.

[0104] A group of ten horses of different ages and sexes participated in this study. The results are shown in... Figures 10-12 In the middle. For example Figure 10 and 12 As shown, on day 3, many horses experienced a 100% decrease in sensitivity by day 10, a phenomenon that persisted until day 90, while other horses showed a continuous improvement in sensitivity throughout the observation period. Similarly, as... Figure 11 and 12 As shown, some horses showed a decrease in joint deformities by day 3, which stabilized and persisted until day 90, while other horses showed continuous improvement throughout the observation period. Figure 12 The chart on the far right shows that all horses experienced a significant reduction in discomfort during movement between the first and last examinations.

[0105] Overall, this study showed that the sensitivity decreased from a mean of 2.92 on day 0 to a mean of 0.67 on day 90, with a 71% decrease on day 10 and continued improvement up to a 77% decrease by day 90. On average, the joint deformity was 3.77 on day 0 and decreased to 1.78 on day 90, indicating that improvement in joint deformity requires a longer time. The improvement in joint deformity was 15% on day 10 and reached 53% on day 90. In summary, this study demonstrates that the cultured equine gingival fibroblast injection according to the present invention has beneficial effects on joint deformities and tenderness in individuals with arthroarthritis, and is well tolerated by the animals.

[0106] While various illustrative embodiments of the invention have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the 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. The cultured mammalian cell population of claim 1, wherein about 10% or less of the cells in the population express at least one mRNA selected from the group consisting 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 mRNA.

3. The cultured mammalian cell population of 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 mRNA.

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 derived from a cultured mammalian cell population according to claim 1 and comprising at least one pharmaceutically or cosmetically acceptable carrier or excipient.

7. A method for culturing a mammalian cell population 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, the method comprising: Obtain tissue containing mammalian gingival fibroblasts; Enzymatic digestion of the tissue produces an aggregate of separated cells; The process of inheritance includes: (i) The collection of the separated cells is cultured for a first time period in a culture medium mixture comprising at least 20% fetal bovine serum and basic fibroblast growth factor to produce adherent cells; (ii) At the end of the first time period, wash the adherent cells and replace the culture medium mixture; as well as (iii) The adherent cells are cultured for a second time period; as well as (iv) At the end of the second time period, the adherent cells are trypsinized to produce a subsequent collection of separated cells, and the subsequent collection of separated cells is washed. as well as Repeat (i) to (iv) to perform multiple generations; as well as At the end of the last of the multiple passages, the adherent cells are trypsinized, washed, and packaged for storage to produce a cultured mammalian cell population.

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

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

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

11. The method of claim 7, wherein during at least one of the multiple passages, the adherent cells proliferate more than tenfold.

12. The method of claim 7, wherein the cultured mammalian cell population is cultured such that about 10% or less of the cells in the population express at least one mRNA selected from the group consisting 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 mRNA.

13. The method of 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 mRNA.

14. The method of claim 7, wherein the mammalian gingival fibroblasts are allogeneic.

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

16. A method of treating a mammalian ailment with an agent comprising or derived from a cultured mammalian cell population containing 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, the method comprising applying the agent topically to the mammal or injecting the agent into the mammal.

17. The method of claim 16, wherein the ailment is a skin ailment selected from the group consisting of: skin wounds, skin aging, hair loss, allergic reactions, atopic dermatitis, asthma, or pressure sores, and wherein applying the agent topically to the mammal further comprises applying the agent topically to the area near the skin ailment of the mammal.

18. The method of claim 16, wherein the ailment is an orthopedic condition selected from the group consisting of: arthropathy, joint degeneration, osteoarthritis, cartilage defects, and tendinitis, and wherein injecting the agent into the mammal comprises injecting a dose between 10 million and 30 million cells into the joints or tendons of the mammal.

19. The method of claim 16, further comprising culturing allogeneic mammalian gingival fibroblasts to prepare the agent.

20. The method of claim 16, further comprising culturing autologous mammalian gingival fibroblasts to prepare the agent.

21. The method of claim 16, wherein the ailment is nerve damage, and wherein the agent is injected into the mammal in a dose between 10 million and 30 million cells in the vicinity of the damaged nerve.

22. The method of claim 16, wherein the ailment is a tumor, and wherein injecting the agent into the mammal comprises injecting a dose between 10 million and 30 million cells into the tumor.

23. The method of claim 16, wherein the mammal is selected from the group consisting of: humans, equines, canines, felines, camels, dromedaries, llamas, bovines, capitols, and mustelids.

24. The method of claim 1, wherein the mammalian gingival fibroblasts are selected from the group consisting of: humans, equines, canines, felines, camels, dromedaries, llamas, bovids, capitols, and mustelids.

25. The method of claim 7, wherein the mammalian gingival fibroblasts are selected from the group consisting of: humans, equines, canines, felines, camels, dromedaries, llamas, bovines, capitols, and mustelids.

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