Cell population comprising mesenchymal cells, pharmaceutical composition comprising cell population, exosome obtained from cell population, and method for producing cell population

Abstract

The present invention addresses the problem of providing a cell population isolated from a living body, which contains mesenchymal cells that have a prescribed cell surface antigen molecule and can be used in the production of a cell sheet. A cell population that is isolated from a living body and contains 80% or more of mesenchymal cells that express at least one type of cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 is used. The mesenchymal cells are preselected from cells collected from tissues in the oral cavity or a culture thereof, and preferably express at least three or more types of cell surface antigen molecules selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

Description

Technical Field

[0001] The present invention relates to cell populations comprising mesenchymal cells expressing predetermined cell surface antigen molecules, three-dimensional cell cultures comprising the cell populations, pharmaceutical compositions comprising the cell populations, exosomes obtained from the cell populations, and methods for producing the cell populations. Background Art

[0002] In recent years, cell transplantation therapy has garnered attention as an effective treatment for various diseases and tissue damage. Even when cells are simply transplanted, they have difficulty engrafting in tissues. Therefore, cell transplantation therapy uses a cell sheet, which is a sheet of cultured cells. This cell sheet allows the transplanted cells to be fixed and attached to a specific tissue.

[0003] The inventors of the present application have developed cell sheets for culturing fibroblasts and disclosed: a method for manufacturing a cell sheet comprising a step of culturing for a predetermined period at a predetermined temperature and under low oxygen conditions (see Patent Document 1); a cell sheet comprising peripheral blood mononuclear cells and fibroblasts for use as a transplant material for the treatment of refractory skin ulcers (see Patent Document 2); and a method for producing a laminated cell sheet comprising a step of inoculating predetermined fibroblasts on a culture substrate to which a culture medium has been added and culturing the cells (see Patent Document 3).

[0004] Fibroblasts were used in each of the above-mentioned cell sheets. Fibroblasts are a type of cell that constitutes connective tissue and are considered to be cells that produce dermal components such as collagen, elastin, and hyaluronic acid and are present in the dermis. These fibroblasts are present throughout the body and have a strong proliferation capacity. On the other hand, the current status of research progress on the characteristics of cell surface antigen molecules is not very significant. As cardiac cell culture materials, for example, fibroblasts expressing VCAM-1 (vascular cell adhesion molecule 1: CD106) are disclosed (see Patent Document 4).

[0005] Fibroblasts are cells derived from the mesenchyme. Previously, mesenchymal stem cells have been disclosed: those that highly express at least one cell surface marker selected from the group consisting of EGFR and MIC-AB (see Patent Document 5); and those that express at least one cell surface marker selected from the group consisting of CD201, CD46, CD56, CD147, and CD165 (see Patent Documents 6 and 7).

[0006] Furthermore, as stem cells from the oral cavity or gums, it is disclosed that: in a cell population of fibroblasts from the oral mucosa (mucosa), the CD56 positivity rate is 43% (see non-patent document 1); in stem cells from the gums, the existing MSC cell surface marker is expressed, so the CD105 positivity rate becomes 99% (see non-patent document 2); in stem cells from the gums, CD29, CD44, CD90, and CD105 are all positive, while CD45 and CD106 are negative (see non-patent document 3); in the review of mesenchymal stem cells and progenitor cells from the gums, the expression of cell surface antigen molecules (see non-patent document 4); the CD105 positivity rate of the cell population of mesenchymal stem cells from the gums is 96% (see non-patent document 5).

[0007] On the other hand, in the production of cell sheets, shortening the cell culture time has a significant impact on cost reduction. It is generally believed that the time it takes for a cell to double (doubling time) is 25 to 53 hours (see Non-Patent Document 6).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. 2016 / 043201 Pamphlet

[0011] Patent Document 2: International Publication No. 2016 / 068217 Pamphlet

[0012] Patent Document 3: Japanese Patent Application Laid-Open No. 2019-38

[0013] Patent Document 4: Japanese Patent Application Laid-Open No. 2016-27797

[0014] Patent Document 5: International Publication No. 2017 / 170925 Pamphlet

[0015] Patent Document 6: International Publication No. 2017 / 188403 Pamphlet

[0016] Patent Document 7: Japanese Patent Application Laid-Open No. 2022-120128

[0017] Non-patent literature

[0018] Non-patent literature 1: Higa, K. et al. Future Sci. OA (2017) 3(4), FSO243

[0019] Non-patent literature 2: Lingqian Du et al. Journal of Dental Sciences (2016) 11, 304e314

[0020] Non-patent literature 3: Ihsene Taihi et al. Stem Cell Research & Therapy (2022) 13:125

[0021] Non-Patent Literature 4: Karim M. Fawzy El-Sayed et al. Stem Cells International Volume 2016, Article ID 7154327, 16 pages.

[0022] Non-patent literature 5: Tomas I Mitrano et al. J Periodontol. 2010 Jun;81(6):917-25.

[0023] Non-patent document 6: Endo Tomohiko et al., Journal of Dental Health (1995) 45, 322-333 Summary of the Invention

[0024] (1) Technical issues to be solved

[0025] The technical problem of the present invention is to provide a cell population isolated from an organism, comprising mesenchymal cells having predetermined cell surface antigen molecules and capable of being used to prepare a cell sheet.

[0026] (2) Technical solution

[0027] The inventors of this application conducted research on allogeneic (allogeneic) cell sheet transplantation for the prevention of postoperative complications and the treatment of intractable skin ulcers. This process focused on the discarded gums removed during wisdom tooth extraction in young people in their teens and twenties. They then analyzed the characteristics of cell surface antigen molecules in mesenchymal cells collected from oral tissue, leading to the development of the present invention.

[0028] That is, the present invention is as follows.

[0029] [1] A cell population isolated from a living organism, comprising 80% or more of mesenchymal cells expressing at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0030] [2] The cell population described in [1] above, wherein the mesenchymal cells are cells collected from oral tissue or a culture thereof.

[0031] [3] The cell population according to [1] or [2] above, wherein the mesenchymal cells express at least three or more cell surface antigen molecules selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0032] [4] The cell population described in [1] or [2] above, comprising 60% or more of mesenchymal cells expressing CD274.

[0033] [5] The cell population described in [1] or [2] above, comprising less than 5% of mesenchymal cells expressing SSEA-3.

[0034] [6] A three-dimensional cell culture comprising the cell population described in [1] or [2] above.

[0035] [7] A pharmaceutical composition comprising the cell population described in [1] or [2] above.

[0036] [8] The pharmaceutical composition according to [7] above, further comprising a pharmaceutically acceptable additive.

[0037] [9] The pharmaceutical composition according to [7] or [8] above, which is in the form of a three-dimensional cell culture.

[0038]

[10] The pharmaceutical composition according to [7] or [8] above, which is in the form of a cell sheet.

[0039]

[11] Exosomes obtained from the cell population described in [1] or [2] above.

[0040]

[12] A method for producing the cell population described in [1] or [2] above, comprising:

[0041] (a) a step of collecting mesenchymal cells from a living tissue, and (b) a step of culturing the collected mesenchymal cells.

[0042]

[13] The method according to

[12] , further comprising, after step (b):

[0043] (c) a step of confirming the expression of the cell surface antigen molecule using an antibody that recognizes at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0044]

[14] The method according to

[12] or

[13] above, wherein in step (b), the culture is carried out using a culture medium containing inflammatory cytokines.

[0045]

[15] The method according to

[12] or

[13] , further comprising, after step (b):

[0046] (d) A step of cryopreserving the cultured cells.

[0047] Furthermore, another embodiment of the present invention is as follows.

[0048] [1'] A cell population isolated from a living organism, comprising 80% or more of mesenchymal cells expressing at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0049] [2'] The cell population described in [1'] above, wherein the mesenchymal cells are cells collected from oral tissue or a culture thereof.

[0050] [3'] The cell population according to [1'] or [2'] above, wherein the mesenchymal cells express at least three or more cell surface antigen molecules selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0051] [4'] The cell population according to any one of [1'] to [3'] above, comprising 60% or more of mesenchymal cells expressing CD274.

[0052] [5'] The cell population according to any one of [1'] to [4'] above, comprising less than 5% of mesenchymal cells expressing SSEA-3.

[0053] [6'] A three-dimensional cell culture comprising the cell population described in any one of [1'] to [5'] above.

[0054] [7'] A pharmaceutical composition comprising the cell population described in any one of [1'] to [5'] above.

[0055] [8'] The pharmaceutical composition according to [7'] above, further comprising a pharmaceutically acceptable additive.

[0056] [9'] The pharmaceutical composition according to [7'] or [8'] above, which is in the form of a three-dimensional cell culture.

[0057] [10'] The pharmaceutical composition according to any one of [7'] to [9'] above, which is in the form of a cell sheet.

[0058] [11'] Exosomes obtained from the cell population described in any one of [1'] to [5'] above.

[0059] [12'] A method for producing a cell population as described in any one of [1'] to [5'] above, comprising:

[0060] (a) a step of collecting mesenchymal cells from a living tissue, and (b) a step of culturing the collected mesenchymal cells.

[0061] [13'] The method as described in [12'] above, which further comprises, after step (b):

[0062] (c) a step of confirming the expression of the cell surface antigen molecule using an antibody that recognizes at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0063] [14'] The method according to [12'] or [13'] above, characterized in that in step (b), the culture is carried out using a culture medium containing inflammatory cytokines.

[0064] [15'] The method according to any one of [12'] to [14'] above, further comprising, after step (b):

[0065] (d) A process for freezing and preserving cells cultured in food.

[0066] Furthermore, another embodiment 2 of the present invention is as follows.

[0067]

[16] A method for preventing or treating ischemic tissue, stubborn skin ulcers, burns, ischemic heart disease, severe limb ischemia or postoperative complications, characterized in that the pharmaceutical composition described in any one of [7'] to [10'] above is transplanted into a subject requiring transplantation.

[0068]

[17] Use of the cell population described in any one of [1'] to [5'] above in the manufacture of a pharmaceutical composition for preventing or treating ischemic tissue, stubborn skin ulcers, burns, ischemic heart disease, critical limb ischemia or postoperative complications.

[0069] (3) Beneficial effects

[0070] The cell populations of the present invention can be used for cell transplantation therapy or to produce cell sheets for use in such treatments. Furthermore, by culturing mesenchymal cells collected from the gums to produce a cell population, these cells can be easily collected from the gums of healthy individuals, for example, during dental treatment. Furthermore, the cell populations of the present invention exhibit rapid cell proliferation, enabling rapid production of cell sheets. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This figure shows a photograph of the cell sheet produced in Example 5.

[0072] Figure 2 This is a graph showing the results of examining the secretion of vascular endothelial growth factor (VEGF) from a cell sheet in Example 8.

[0073] Figure 3 This is a graph showing the results of examining the expression of SSEA-3 in gingival mesenchymal cells in Example 9. DETAILED DESCRIPTION

[0074] <Cell Population>

[0075] The cell population of the present invention is not particularly limited as long as it is a cell population isolated from a living organism and contains more than 80% of mesenchymal cells expressing at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280, and is hereinafter also referred to as the "cell population of the present invention."

[0076] In this specification, "mesenchymal cells" refers to a general term for cells that are present in the mesenchymal tissue that is the connective tissue formed between the ectoderm and the endoderm in embryology, and fibroblasts can be exemplified. In addition, the mesenchymal cells can be listed as mesenchymal cells or cultures thereof collected from oral tissues such as the gums or the lower half of the salivary glands (the inside of the cheek), adipose tissue, bone marrow or skin. In addition, the above-mentioned "cultures thereof" refer to cultured cells of the above-mentioned mesenchymal cells, which are cells that can maintain the expression of cell surface molecules in the above-mentioned mesenchymal cells.

[0077] " fibroblast " in this manual is to help as the cell type of the connective tissue of the tissue or organ that maintains body and make it to the fibrous material of combination.Do not have significant function particularly in normal tissue, but if damaged, then can wander to the damaged portion, by secreting collagen, elastin or the hyaluronic acid etc. that assist in maintaining the reticular structure of tissue, begin to produce extracellular matrix, have the function of renewing extracellular matrix. In addition, bring out wound contraction etc., in the wound healing process, play an important role. For this fibroblast preparation method, except the method for putting down in writing among the above-mentioned patent documentation 2, also can enumerate the preparation method of mesenchymal cell described later, but be not limited to this, as long as be by the cell mass that obtains of the condition that prepares usually as fibroblast separation in the present technical field.

[0078] The source of mesenchymal cells is not particularly limited. Examples include mammals such as humans, pigs, monkeys, chimpanzees, cows, horses, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats. Furthermore, the term "source" is used to indicate the animal species from which the cells were collected.

[0079] The CD (cluster of differentiation) classification of cell surface antigen molecules in this specification is based on the CD classification listed on the HGNC website (www.genenames.org / data / genegroup / #! / group / 471). In addition, the gene names in this specification are based on the gene names listed on the GeneCards (registered trademark) website (www.genecards.org).

[0080] The cell population of the present invention preferably comprises 80% or more of the aforementioned mesenchymal cells, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. The mesenchymal cells included in the cell population of the present invention may be a single cell surface antigen or a combination of two or more cells, as long as they express at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. That is, the cell population in the present invention may contain, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more of mesenchymal cells expressing only CD13, CD59, CD49e, CD151, or CD280, or may contain 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more of mesenchymal cells expressing two, three, four, or five types selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0081] The mesenchymal cells herein are not particularly limited as long as they express at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. However, they preferably express at least two of the group consisting of CD13, CD59, CD49e, CD151, and CD280, more preferably at least three of the group consisting of CD13, CD59, CD49e, CD151, and CD280, further preferably at least four of the group consisting of CD13, CD59, CD49e, CD151, and CD280, and particularly preferably express CD13, CD59, CD49e, CD151, and CD280.

[0082] When at least two species selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least two species may be any one of the following: CD13 and CD59; CD13 and CD49e; CD13 and CD151; CD13 and CD280; CD59 and CD49e; CD59 and CD151; CD59 and CD280; CD49e and CD151; CD49e and 280; CD151 and CD280.

[0083] When at least three species selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least three species may be any one of the following: CD13, CD59 and CD49e; CD13, CD59 and CD151; CD13, CD59 and CD280; CD13, CD49e and CD151; CD13, CD49e and CD280; CD13, CD151 and CD280; CD59, CD49e and CD151; CD59, CD49e and CD280; CD59, CD151 and CD280; CD49e, CD151 and CD280.

[0084] When at least four species selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least four species may be any one of the following: CD13, CD59, CD49e and CD151; CD13, CD59, CD49e and CD280; CD13, CD59, CD151 and CD280; CD13, CD49e, CD151 and CD280; CD59, CD49e, CD151 and CD280.

[0085] The above-mentioned cell population can further include a cell population containing more than 50%, preferably more than 60%, more preferably more than 70%, further preferably more than 80%, and particularly preferably more than 90% of mesenchymal cells expressing CD29, CD63, CD90, CD73, CD44, CD13 and / or HLA class I.

[0086] Furthermore, the cell population may include a cell population containing 10% or less, preferably 5% or less, more preferably 3% or less, and even more preferably 1% or less of mesenchymal cells expressing CD304, CD106, CD34, and / or CD45.

[0087] Furthermore, the cell population may include one containing 80% or less, preferably 70% or less, and more preferably 15 to 68% of mesenchymal cells expressing CD105.

[0088] In addition, the above-mentioned cell population may include a cell population containing 5% or less, preferably 3% or less, more preferably 1% or less, particularly preferably 0.3% or less, and most preferably 0% of mesenchymal cells expressing SSEA-3 (Stage-Specific Embryonic Antigen-3), a cell surface antigen molecule known as a marker for pluripotent stem cells.

[0089] In addition, when preparing a pharmaceutical composition in the form of a cell sheet containing the above-mentioned cell population for allogeneic transplantation, examples include a cell population containing 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more of mesenchymal cells expressing CD274 in order to suppress immune rejection reactions.

[0090] The expression of the cell surface antigen molecules in cells can be confirmed, for example, using antibodies that can bind to the cell surface antigen molecules. Specifically, taking the expression of CD13 as an example, a suspension of the cell population can be prepared, and the expression of CD13 in the cells constituting the cell population can be confirmed using an anti-CD13 antibody.

[0091] In this specification, "expression" refers to the expression of a protein of a cell surface antigen molecule, but preferably refers to the presentation of a cell surface antigen molecule on the cell surface.

[0092] In addition, in the cell population of the present invention, there is no particular limitation on the cells included other than the above-mentioned mesenchymal cells, but examples thereof include lymphoid cells such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells.

[0093] <Three-dimensional cell culture>

[0094] The three-dimensional cell culture in this specification is not particularly limited as long as it includes the above-mentioned cell group. The form of the three-dimensional cell culture is that cells are physically and functionally connected to each other via adhesion molecules, extracellular matrix, etc., so as to have the form of the target three-dimensional structure. Wherein, although "three-dimensional cell culture" includes cell sheets, spheroids, organoids, etc., it is not limited thereto. As the shape of the three-dimensional cell culture, sheets, organoids, spheroids, tissue-like, hollow or block-like shapes can be listed, but it is not limited thereto. In addition, typically, as cell sheets, attached cells (cells that are attached to a culture substrate and proliferated, such as fibroblasts) cells are easily physically and / or functionally connected to each other and form a cell aggregate of a three-dimensional structure, but for example spheroids, even if they are suspended cells (cells that are suspended in a culture medium and proliferated, such as hematopoietic stem cells, blood cells), when cells are physically and / or functionally connected to each other and form a cell aggregate of a three-dimensional structure, they also belong to the three-dimensional cell culture in this specification.

[0095] The method for producing a three-dimensional cell culture can be performed using methods known to those skilled in the art and is not particularly limited. Examples of methods for producing a three-dimensional cell culture include those disclosed in Japanese Patent Application Laid-Open Nos. 2012-120696, 2017-176025, and 2015-149905.

[0096] The thickness of the three-dimensional cell culture is not particularly limited and may be, for example, 0.01 mm to 10 mm, 0.1 mm to 5 mm, 0.5 mm to 4 mm, or 1.0 mm to 3 mm.

[0097] In a three-dimensional culture containing a cell population, the density of the cells cultured in the three-dimensional culture is, for example, 5×10 1 cells / cm 3 Above and 5×10 10 cells / cm 3 More preferably 5×10 2 cells / cm 3 Above and 5×10 9 cells / cm 3 Below, more preferably 5×10 3 cells / cm 3 Above and 1×10 9 cells / cm 3 Below, particularly preferably 5×10 4 cells / cm 3 Above and 5×10 8 cells / cm 3 the following.

[0098] <Pharmaceutical Composition>

[0099] The pharmaceutical composition of the present invention is not particularly limited as long as it comprises the above-described cell population and is hereinafter referred to as the "pharmaceutical composition of the present invention." The pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of the form of the pharmaceutical composition of the present invention include a suspension form and a three-dimensional cell culture form, with the cell sheet form being preferred.

[0100] The present pharmaceutical composition is preferably a pharmaceutical composition for the treatment of ischemic tissue. Wherein, "ischemic tissue" refers to a tissue in a state of decreased blood flow. In ischemic tissue, tissue collapse occurs as blood flow decreases. The present pharmaceutical composition promotes angiogenesis in such ischemic tissue by secreting VEGF, thereby improving blood flow. As target diseases for treatment, refractory skin ulcers represented by bedsores, occlusive arteriosclerosis, diabetes, venous insufficiency, collagen disease, vasculitis, etc. can be listed. In addition, when using the present pharmaceutical composition for the prevention or treatment of refractory skin ulcers, the present pharmaceutical composition can be transplanted to an object in need of transplantation, such as a patient with refractory skin ulcers. In addition, as target diseases for treatment, ischemic tissue, burns, ischemic heart disease, severe limb ischemia, and postoperative complications (such as incomplete suture of various organs, bronchial fistula, pancreatic fistula, and bile leakage) can be listed.

[0101] Furthermore, the pharmaceutical composition of the present invention can be used for the treatment of refractory skin ulcers such as ischemic ulcers, ulcers associated with collagen diseases, and ulcers associated with radiation therapy, or for the prevention of postoperative complications, and can also be used as an enhancer for such prevention.

[0102] In this specification, "pharmaceutically acceptable additives" include physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers and lubricants.

[0103] The cell sheet morphology in the pharmaceutical composition of the present invention is a morphology in which cells are physically or functionally connected to each other directly or via viscosity molecules, extracellular matrix, etc., to form a sheet structure. The method for producing a cell sheet can be carried out by methods known to those skilled in the art and is not limited thereto. Examples of methods for producing a cell sheet include the methods taught in the aforementioned Patent Documents 1 to 3, Japanese Patent Application Publication No. 2011-006490, International Patent Publication No. 2015 / 068505, Japanese Patent Application Publication No. 2022-8269, and the like.

[0104] The term "cell sheet" as used herein may be a single-layer structure consisting of one cell layer or a laminated structure consisting of two or more cell layers. The laminated structure is not particularly limited, and may include multilayer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0105] The thickness of the cell sheet as used herein is not particularly limited. Examples of the thickness of the cell sheet include, for example, 0.001 mm to 2.0 mm. More preferably, it is 0.01 mm to 1.5 mm, even more preferably 0.03 mm to 1.2 mm, and particularly preferably 0.05 mm to 1.0 mm. By setting the thickness of the cell sheet within this range, excellent shape-retention properties can be achieved, which contributes to high cell survival rates within the cell sheet and facilitates cell transplantation.

[0106] The suspension form in this specification refers to a state in which cells are suspended in a solution such as a culture medium, PBS, or physiological saline, either alone or in contact with other cells.

[0107] <Exosomes>

[0108] The exosomes of this specification are not particularly limited as long as they are obtained from the above-mentioned cell population of this invention, and are hereinafter referred to as "exosomes of this invention". In addition, in this specification, "exosomes" refer to vesicles composed of a membranous double membrane from cells. Almost all cell types release exosomes, which can be found in many body fluids and contain nucleic acids or proteins such as DNA, mRNA or microRNA. In a preferred embodiment, the exosomes of this specification are exosomes obtained from the cell population of this invention comprising mesenchymal cells collected from human gums. Exosomes obtained from the cell population of this invention are preferred in the following aspects: they have anti-inflammatory effects and promote wound healing effects; since mesenchymal cells can be cultured in a serum-free environment, exosomes are easy to recover; and since mesenchymal cells have almost no risk of canceration, exosomes are highly safe and contain various growth factors. As the size of the exosomes of this invention, for example, a diameter of 20nm to 140nm can be listed.

[0109] The method for producing exosomes in the present invention can be performed by removing cells and contaminants from the culture supernatant of the cell population in the present invention using a centrifuge or ultrafiltration and recovering the supernatant. The above-mentioned centrifugation can be performed, for example, at 1000 rpm to 3000 rpm for 2 to 10 minutes. Subsequently, further purified exosomes can be recovered using a commercially available kit for isolating exosomes. Exosomes can also be purified / recovered by ultracentrifugation (e.g., 100,000 g to 1,000,000 g for 30 minutes to 12 hours).

[0110] <Method for Producing Cell Populations>

[0111] The method for producing the cell population of this specification is not particularly limited as long as it sequentially includes the steps of (a) collecting mesenchymal cells from biological tissue and (b) culturing the collected mesenchymal cells, and is hereinafter referred to as the "method for producing the cell population of this case."

[0112] After operation (b), can also comprise (c) use the antibody of at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280, confirm the operation of the expression of this cell surface antigen molecule, but be not limited to this.In addition, after operation (b), can also comprise (d) the operation of the cell freezing preservation that is cultivated, but be not limited to this.About the time of freezing preservation and freezing preservation method, will be described later.

[0113] The method for culturing cells is not particularly limited, and conventional methods used in the fields of regenerative medicine, pharmaceuticals, quasi-drugs, etc. can be used. Specifically, for example, a method in which a culture medium is added to a culture vessel and the cells to be cultured are seeded and cultured under an optimal environment until the desired state is achieved is mentioned.

[0114] The culture medium used in the cell culture method is a solution containing components required for cell culture. The culture medium is not particularly limited as long as it is suitable for culturing the cells. Examples of culture medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, and additives.

[0115] Furthermore, these culture solution components may be blended alone or in combination of two or more. These culture solution components may be appropriately adjusted according to the cells to be cultured, etc., from known culture solution components.

[0116] Examples of the sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, oligolacto-fructose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0117] Examples of the amino acids include L-glutamic acid, L-glutamine, L-arginine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0118] Examples of vitamins include sodium ascorbate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0119] Examples of the inorganic salt include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium hydrogen carbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium hydrogen carbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate.

[0120] Examples of the trace metal include iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0121] Examples of additives include serum such as fetal bovine serum, horse serum, or human serum; growth factors such as FGF2, EGF, HGF, VEGF, or PDGF; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, or ascorbic acid derivatives; antibiotics such as penicillin or streptomycin; pH adjusters such as HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid); organic acids such as lactic acid or propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine or putrescine; reducing agents such as mercaptoethanol or 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, or pullulan; Rho kinase inhibitors, etc.

[0122] Furthermore, when the cultured cells are stem cells or progenitor cells, differentiation-inducing factors may be added to the culture medium to induce differentiation during the culture. Examples of differentiation-inducing factors include activin A, BMP4, bFGF, VEGF, SCF, DKK1, BMP signaling inhibitors, TGFβ / activin / NODAL signaling inhibitors, Wnt signaling inhibitors, and retinoic acid signaling inhibitors.

[0123] In addition, in the above step (b), culture can also be carried out using a culture medium containing inflammatory cytokines such as IFN-γ, interleukin-17 (IL-17), tumor necrosis factor-α (TNF-alpha (TNF-α)), IL-1, IL-2, IL-12, and IL-18.

[0124] In step (c), the expression of the cell surface antigen molecule can be confirmed using an antibody that recognizes at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280, but the expression of each cell surface molecule can also be confirmed using an antibody that recognizes CD13, CD59, CD49e, CD151, or CD280. Further, the proportion (%) of cells expressing each cell surface molecule in the cell population can be confirmed by counting the number of cells constituting the cell population and the number of cells expressing each cell surface molecule. In addition, whether it is a mesenchymal cell can be confirmed by using an antibody that recognizes CD90.

[0125] Specific examples of the culture medium containing the above-mentioned culture medium components include AIM (registered trademark) V medium, HFDM-1 medium, balanced buffers such as Dulbecco's phosphate buffered saline (D-PBS) or Hank's balanced salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 medium, Williams' medium E, Brinster's BMOC-3 medium, E8 medium and other culture media.

[0126] The culture conditions are not particularly limited as long as they can bring the cultured cells to the target state. As general culture conditions, for example, an adjusted basal culture medium is used at 37°C and 5% CO2. The culture conditions can be appropriately set according to the cells to be cultured, etc. In addition, there are no particular restrictions on the time of cell culture as long as the cultured cells are formed into the target state. As for the time of cell culture, for example, it is within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, or within 3 days.

[0127] The number of cells seeded in the culture container is not particularly limited as long as it is suitable for the cells to be cultured and the culture container. For example, the number of cells seeded in the culture container is 1×10 3 cells / mL and above 1×10 7 Cells / mL or less. More preferably 1×10 4 cells / mL and above 1×10 6 cells / mL or less, more preferably 5×10 4 cells / mL and above 5×10 6 cells / mL or less, particularly preferably 1×10 5 cells / mL and above 1×10 6 cells / mL or less.

[0128] The density of the cultured cells is not particularly limited as long as it is suitable for the cultured cells, the culture container, and the purpose for which the cells are cultured. For example, the planar density of the cultured cells is 1×10 4 cells / cm 2 Above and 1×10 9 cells / cm 2 More preferably 1×10 5 cells / cm 2 Above and 1×10 8 cells / cm 2 Below, more preferably 2×10 5 cells / cm 2 Above and 1×10 7 cells / cm 2 Below, particularly preferably 5×10 5 cells / cm 2 Above and 1×10 6 cells / cm 2 the following.

[0129] When a pharmaceutical composition containing a cell population is prepared as a cell sheet or a three-dimensional culture containing the cell population, the density of the cells in the cultured cell sheet or three-dimensional culture is, for example, 5×10 1 cells / cm 3 Above and 5×10 10 cells / cm 3 More preferably 5×10 2 cells / cm 3 Above and 5×10 9 cells / cm 3 Below, more preferably 5×10 3 cells / cm 3 Above and 1×10 9 cells / cm3 Below, particularly preferably 5×10 4 cells / cm 3 Above and 5×10 8 cells / cm 3 the following.

[0130] The mesenchymal cells herein may be autologous cells derived from the patient to whom the pharmaceutical composition is to be administered, or may be allogeneic cells derived from another subject of the same species, for example, another patient.

[0131] The dosage of the pharmaceutical composition of the present invention is, when in a suspended state, 5×10 2 ~1×10 12 pcs / time, preferably 1×10 4 ~1×10 11 pcs / time, more preferably 1×10 5 ~1×10 10 In addition, this dosage can be taken as a single dose and administered multiple times, or it can be divided into multiple doses. In addition, usually, when administered to adults, the number of cells per unit body weight is 1×10~5×10 10 / kg, preferably 1×10 2 ~5×10 9 / kg, more preferably 1×10 3 ~5×10 8 In addition, the present dosage may be administered as a single dose or divided into multiple doses.

[0132] <Freezing>

[0133] The cell population, the three-dimensional cell culture, the pharmaceutical composition and the exosomes of this case can be frozen and stored. Specifically, they can be frozen and stored at -20°C or below, preferably at -80°C for more than 1 day, more than 1 week and less than 10 years, more than 1 month and less than 5 years, more than 1 year and less than 3 years. As freezing methods, methods for freezing using cell cryopreservation solutions widely used in bioengineering, freezing methods described in Japanese Patent Laid-Open No. 2022-8269, slow freezing methods or vitrification methods can be listed. The above-mentioned freezing time and freezing method, in the above-mentioned method for making a cell population, are applicable to after step (b), and can also be applied to the step (d) of freezing and storing the cultured cells.

[0134] The cell population of the present case that has been cryopreserved can be stored in a cell bank. Taking the case of collecting mesenchymal cells from gingival tissue as an example, specifically, when a patient who has given consent has their wisdom teeth extracted, mesenchymal cells are collected from the gingival tissue excised during subsequent suturing and cultured (P0). Next, the collected mesenchymal cells (P0) are inoculated, the culture medium is exchanged 1 hour later, and the adhered cells are cultured. The cells (P1) obtained by detaching the cultured cells from the culture medium can be cryopreserved and stored in the bank.

[0135] When applied to regenerative medicine, for example, the cryopreserved cells (P1) stored in the above-mentioned bank are inoculated on a culture dish and cultured to P4 and then cryopreserved. They can be thawed and used for transplantation when needed. In addition, the numbers in P0, P1, and P4 above represent the passage numbers.

[0136] Examples

[0137] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited by these exemplifications.

[0138] [Example 1] Preparation of mesenchymal cells collected from gingival tissue

[0139] First, cells from the gingiva were cultured using the following method to prepare P1 cells, P2 cells, or P3 cells for use in the subsequent examples. In addition, the numbers in P1, P2, and P3 above represent the passage numbers.

[0140] <Preparation of P1 cells>

[0141] · The gingival tissue of 5 donors (No1 - 5) was minced with forceps and a scalpel, and the minced gingival tissue was attached to a 6-well plate (#MS-80060S: Sumitomo Bakelite Co., Ltd.).

[0142] · 1.5 mL of fetal bovine serum NeoSERA (registered trademark) (Biomedical Japan Inc.), 15 mL of AIM-V medium (Thermo Fisher Scientific), and 15 mL of HFDM-1(+) (Cell Science & Technology Institute, Inc.) were added to one 50 mL tube, and the liquid was aspirated and discharged to adjust the culture medium.

[0143] · 5 mL of this culture medium was added to each well of the 6-well plate, and the minced gingival tissue was cultured. The cells P0 that grew from the minced gingival tissue were collected, the cells were inoculated on a 90 mm culture dish (#MS-13900S: Sumitomo Bakelite Co., Ltd.), the culture medium was exchanged 1 hour later, and the adhered cells were cultured.

[0144] · One piece of culture medium for a 90 mm petri dish is 4.5 mL of AIM-V medium, 4.5 mL of HFDM-1(+), and 1 mL of NeoSERA.

[0145] · When the density of the cells cultured in the 90 mm petri dish reaches 70 - 95%, P1 cells are recovered. Using STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: ZENIAQ Resource Co., LTD.), the P1 cells are adjusted to 1×10 6 cells / mL, and dispensed into cryotubes at a rate of 1 mL each to prepare a cell preservation solution containing P1 cells, which is cryopreserved at -80 °C until use.

[0146] <Expression of CD90 in P1 cells>

[0147] The expression of CD90 in P1 cells is confirmed by the following method.

[0148] · Adjust P1 cells to 1×10 6 cells / mL using PBS, and place the 15 mL tube on ice.

[0149] · Put 20 μL of the anti-CD90 antibody or isotype (ISO) control antibody shown in Table 1 into each 1.5 mL tube, then add 100 μL of the above P1 cells (1×10 6 cells / mL), aspirate and dispense the liquid, cover it from light on ice, and let it stand for 30 minutes. In Table 1, ISO represents the isotype control.

[0150] · Add 400 μL of PBS to the 1.5 mL tube, aspirate and dispense the liquid, and transfer it to a 5 mL polystyrene round-bottom tube (#REF352235, Becton Dickinson) with a cell strainer cap.

[0151] · Transfer it to a PP standard test tube (round bottom) (#A26428, Beckman Coulter), and place it on ice until the sample measurement starts.

[0152] · For No.1, measure the fluorescence wavelength using Novocyte (ACEA Biosciences), and for No.2 - 5, measure the fluorescence wavelength using Cytomics FC500 (Beckman Coulter).

[0153] [Table 1]

[0154]

[0155] The results are shown in Table 2. From the results in Table 2, it can be judged that the obtained cell population contains more than 95% of CD90, which is a marker of mesenchymal cells, and most of the obtained P1 cell population are mesenchymal cells. In addition, when the minced gingival tissue at the start of culture was set as day 0, for the viable cell count of P1 cells on day 13 of culture, No. 1 to 5 were 13.8×10 6 cells / mL, 43.1×10 6 cells / mL, 45.6×10 6 cells / mL, 37×10 6 cells / mL, 22×10 6 cells / mL, respectively.

[0156] [Table 2]

[0157]

[0158] [Preparation of P2 cells]

[0159] The preparation of P2 cells is carried out through the following procedures.

[0160] · Thaw the cell preservation solution containing P1 cells dispensed into the above cryotubes.

[0161] · Add 3.5 mL of AIM-V medium (Thermo Fisher Scientific), 0.5 mL of bovine serum for cell culture NeoSERA (registered trademark) (Biomedical Japan Inc.), and 1 mL of the thawed cell preservation solution containing P1 cells to a 15 mL tube, and centrifuge at 1200 rpm for 1 minute at room temperature.

[0162] · Aspirate and remove the supernatant, and inoculate the P1 cells onto two 10 cm culture dishes (#93100: BM Equipment Co., Ltd.) and culture in an incubator (37°C, 5% CO2) for 4 days.

[0163] The medium for the 10 cm culture dish uses 5 mL of AIM-V medium + HFDM-1(+) (Cell Science & Technology Institute, Inc.) + 1 mL of NeoSERA.

[0164] · Aspirate and remove the culture solution from the 10 cm culture dish, add 5 mL of PBS (Cell Science & Technology Institute, Inc.) to one 10 cm culture dish, wash the inside of the culture dish, and then aspirate and remove the PBS.

[0165] · Add 2 mL of r-TE (Cell Science & Technology Institute, Inc.), a recombinant trypsin / EDTA solution, to one 10-cm Petri dish and leave the 10-cm Petri dish in an incubator (37 °C, 5% CO₂) for 3 minutes.

[0166] · Install a 40-μm cell strainer (#352340: Corning) in a 50-mL tube.

[0167] · Detach the cells from the 10-cm Petri dish by aspirating and discharging, and transfer them to the above 50-mL tube.

[0168] · Add 2 mL of s-TI (Cell Science & Technology Institute, Inc.), a synthetic trypsin neutralizer solution, to one 10-cm Petri dish, wash the inside of the 10-cm Petri dish, and transfer the washing solution to the above 50-mL tube.

[0169] · Centrifuge the above 50-mL tube at 1200 rpm for 1 minute at room temperature.

[0170] · After centrifugation, aspirate and remove the supernatant, and resuspend the cells in 2 mL of AIM-V medium to obtain a cell suspension containing P2 cells. Mix 10 μL of this cell suspension with 10 μL of trypan blue, spread 10 μL of the mixture on a hemocytometer, and count the number of cells.

[0171] · For the doubling experiment described above, inoculate 2.5 × 10 2 cells of P2 into three cell culture flasks (#MS-23800 225 cm 5 ²: Sumitomo Bakelite Co., Ltd.). Use 25 mL of AIM-V medium + 25 mL of HFDM-1(+) + 2 mL of NeoSERA for the culture medium of one cell culture flask.

[0172] · Adjust the remaining cells to 1 × 10 6 cells / mL of P2 cells using STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: ZENIAQ Resource Co., LTD.), prepare a cell preservation solution containing P2 cells by dispensing 1 mL into each cryotube, and freeze and store at -80 °C until use.

[0173] <Preparation of P3 cells>

[0174] · Passage the P2 cells inoculated into the cell culture flask on the 3rd day for No. 1, and passage the P2 cells inoculated into the cell culture flask on the 5th day for No. 2 - 5.

[0175] For No. 2 to No. 5, half of the medium was exchanged 3 days after cell inoculation.

[0176] The culture medium was removed from one cell culture flask by aspiration, and 25 mL of PBS was added to another cell culture flask to wash the cell culture flask, and then the PBS was removed by aspiration.

[0177] · Add 5 mL of recombinant trypsin / EDTA solution (r-TE) (Cell Science & Technology Institute, Inc.) to one cell culture flask, and place the cell culture flask in an incubator (37°C, 5% CO2) for 3 minutes.

[0178] A 40 μm cell strainer (#352340: Corning Incorporated) was installed in a 50 mL tube.

[0179] Remove the cells from the cell culture flask by pipetting and transfer to a 50 mL tube.

[0180] 5 mL of s-TI (Cell Science & Technology Institute, Inc.), a synthetic trypsin neutralizer solution, was added to one cell culture flask, and the cell culture flask was rinsed and the rinse solution was transferred to a 50 mL tube.

[0181] • Centrifuge the 50 mL tube at 1200 rpm for 2 minutes at room temperature.

[0182] After centrifugation, the supernatant was removed by aspiration and the cells were suspended in 20 mL of AIM-V medium to obtain a P3 cell suspension. 10 μL of this cell suspension was mixed with 10 μL of trypan blue and 10 μL of the mixture was applied to a hemocytometer to count the cells.

[0183] For doubling experiments, add 3 cell culture flasks (#MS-23800 225cm 2 :Sumitomo Bakelite Co., Ltd.) were inoculated with 2.5×10 P3 cells 5 cells.

[0184] P3 cells were adjusted to 1×10 using STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: ZENIAQ Resource Co., LTD.). 6 Cell preservation solution containing P3 cells was prepared by aliquoting 1 mL into each cryovial and storing at -80°C until use.

[0185] [Example 2] Expression of cell surface antigen molecules in mesenchymal cells collected from gingival tissue

[0186] Among a large number of cell surface antigen molecules, the inventors of the present application, based on existing knowledge and technical information, focused on CD13, CD29, CD59, CD49e, CD151, CD280, CD304, CD274, CD63, and CD106, or HLA class I and HLA class II (HLA class II), and examined the expression of cell surface molecules known as mesenchymal stem cell markers and these cell surface antigen molecules using antibodies.

[0187] One cryotube prepared in Example 1 (containing the cell preservation solution of P3 cells) was taken out from -80°C and thawed.

[0188] To a 15 mL tube, 3.5 mL of AIM-V medium (Thermo Fisher Scientific), 0.5 mL of bovine cell culture serum NeoSERA (registered trademark) (Biomedical Japan Inc.), and 1 mL of cell preservation solution containing thawed P3 cells were added, and the tube was centrifuged at 1200 rpm for 1 minute at room temperature.

[0189] The supernatant was removed by aspiration and P3 cells were seeded onto two 10 cm culture dishes. Each 10 cm dish was plated with 5 mL of AIM-V medium, HFDM-1(+) (Cell Science & Technology Institute, Inc.), and 1 mL of NeoSERA.

[0190] Culture the 10 cm culture dish in an incubator (37°C, 5% CO2) for 4 days.

[0191] The culture medium was removed by aspiration from the 10 cm culture dish, and 5 mL of PBS (Cell Science & Technology Institute, Inc.) was added to one 10 cm culture dish to wash the dish. The PBS was then removed by aspiration.

[0192] · Add 2 mL of recombinant trypsin / EDTA solution (r-TE) (Cell Science & Technology Institute, Inc.) to a 10 cm culture dish and place the 10 cm dish in an incubator (37°C, 5% CO2) for 3 minutes.

[0193] A 40 μm cell strainer (#352340: Corning Incorporated) was installed in a 50 mL tube.

[0194] Remove the cells from the 10 cm culture dish by pipetting and transfer to a 50 mL tube.

[0195] · Add 2 mL of s-TI (Cell Science & Technology Institute, Inc.), a synthetic trypsin neutralizer solution, to a 10 cm dish, rinse the inside of the 10 cm dish, and transfer the rinse solution to a 50 mL tube.

[0196] • Centrifuge the 50 mL tube at 1200 rpm for 1 minute at room temperature.

[0197] After centrifugation, the supernatant was removed by aspiration and the cells were suspended in 2 mL of PBS to prepare a suspension containing P4 cells. 10 μL of this cell suspension was mixed with 10 μL of trypan blue and 10 μL of the mixture was applied to a hemocytometer to count the cells.

[0198] To make the cell concentration 1×10 6 After adjusting the concentration to 10 cells / mL with PBS, the 50 mL tube was placed on ice.

[0199] · Add 20 μL of the antibodies listed in Table 3 to each 1.5 mL tube, and then add the cell suspension (1×10 6 The cell suspension was aspirated and 100 μL of the suspension was placed on ice in the dark for 30 minutes.

[0200] 400 μL of PBS was added to a 1.5 mL tube, and the resulting solution was pipetted and transferred to a 5 mL polystyrene round-bottom tube with a cell strainer cap (#REF352235: Becton Dickinson).

[0201] Transfer to a PP standard test tube (round bottom) (#A26428: Beckman Coulter) and place on ice until the start of sample measurement.

[0202] The fluorescence wavelength was measured using Cytomics FC 500 (Beckman Coulter).

[0203] [Table 3]

[0204]

[0205] The results are shown in Table 4. As can be seen from Table 4, in the cell populations obtained by culturing any of the mesenchymal cells from gingival tissue No. 1 to 5, more than 95% of cells expressing CD13, CD29, CD59, CD49e, CD151 or CD280 were confirmed. In addition, in the cell populations obtained by culturing any of the mesenchymal cells from gingival tissue No. 1 to 5, more than 97% of cells expressing CD90 or CD73 as markers of mesenchymal stem cells were contained. Furthermore, the above cell populations contained more than 60% of cells expressing CD274 and 16 to 66% of cells expressing CD105. On the other hand, in any of the above cell populations, the expression of CD34, CD45, CD56, CD304, and CD106 was not confirmed, and the cells expressing HLA class II only contained 0 to 0.4%. Furthermore, although the expression of CD13, CD59, CD49e, CD151, and CD280 has not been confirmed in gingival-derived cell populations, novel cell surface molecules have been confirmed in gingival-derived mesenchymal cells.

[0206] [Table 4]

[0207]

[0208] [Example 3] Cell proliferation ability

[0209] To evaluate cell proliferation, we performed an experiment to measure the doubling time. The doubling time here refers to the time it takes for a cell to double in size, that is, the time it takes for one cell to divide and become two.

[0210] The cell doubling time was determined by measuring the number of cells per passage using the following method.

[0211] As described in Example 1 above, 3 cell culture flasks (#MS-23800 225cm 2 : Sumitomo Bakelite Co., Ltd.) were inoculated with 2.5×10 P2 cells prepared in Example 1. 5 After 5 days (2 days after the culture medium was exchanged), the cells were collected and the cell number was counted (P3 doubling experiment).

[0212] Similarly, as described in Example 1 above, 3 cell culture flasks (#MS-23800 225cm 2 : Sumitomo Bakelite Co., Ltd.) were inoculated with 2.5×10 P3 cells prepared in Example 1. 5 cells, and 5 days later (2 days after the medium exchange), the cells were collected and the cell number was counted (P4 doubling experiment).

[0213] Next, subculture was performed by the same method as in Example 1 and the above description, and the number of cells was counted to obtain passage cells (P5 cells to P10 cells). The cell number was confirmed and the doubling time was measured.

[0214] The results are shown in Table 5. The numbers in Table 5 are doubling times (hr), and the upper row's "P3" represents the doubling time from P2 to P3 ("P4" is the doubling time from P3 to P4: P5~P10 is also the same). The doubling times of No. 2~5 are all within 35 hours, and particularly P3, P4 and P5 are within 26 hours. According to Table 1 of above-mentioned non-patent literature 6, the doubling time (doubling time) of cells is generally 25~53 hours. Therefore, it is confirmed that the cell populations of above-mentioned No. 2~5 proliferated in a very short time such as 19~35 hours when the doubling time was 19~35 hours.

[0215] [Table 5]

[0216] Doubling time (hr)

[0217]

[0218] [Example 4] Cell survival rate

[0219] In cell transplantation therapy, it is envisaged that individually separated cells will be cryopreserved and the cells thawed will be used. Thus, it is necessary to highly maintain the survival rate of the cells cryopreserved. Therefore, the cell population obtained by culturing the mesenchymal cells (4 samples) collected from the oral tissue is used to investigate the cell survival rate.

[0220] Approximately 200 mL of blood was collected from patients (aged 40 to 80) with intractable skin ulcers due to venous congestion, and autologous serum was prepared using CELLAID (for serum collection, #JB-SB200CSA: JMS Co., Ltd.).

[0221] Cells were collected from the lower half of the salivary gland (inner cheek) of a patient with a stubborn skin ulcer caused by venous congestion. The tissue was finely minced using forceps and a scalpel, and the minced gingival tissue was attached to a 6-well plate (#MS-80060S: Sumitomo Bakelite Co., Ltd.).

[0222] To a 50-mL tube, add 1.3 mL of autologous serum, 13 mL of AIM-V medium (Thermo Fisher Scientific), 13 mL of HFDM-1(+) (Cell Science & Technology Institute, Inc.), 130 μL of Meiji Collagenase G (#COLGS: Meiji Seika Pharma Co., Ltd.), and 52 μL of Meiji Collagenase H (#COLHS: Meiji Seika Pharma Co., Ltd.), and adjust the culture medium by pipetting.

[0223] · Add 2 mL of this culture medium to each well of a 6-well plate and incubate overnight.

[0224] To a 50 mL tube, add 1.5 mL of autologous serum, 15 mL of AIM-V medium (Thermo Fisher Scientific), and 15 mL of HFDM-1(+) (Cell Science & Technology Institute, Inc.), and adjust the culture medium by pipetting.

[0225] The culture medium was removed from the wells of a 6-well plate and 2 mL of this medium was added to each well of the 6-well plate. The minced lower half of the salivary gland (inner cheek) tissue was cultured. Cells P0 generated from the minced tissue were recovered and seeded onto 90 mm culture dishes (#MS-13900S: Sumitomo Bakelite Co., Ltd.). After 1 hour, the medium was exchanged and the adherent cells were cultured. The culture medium for each 90 mm culture dish consisted of 4.45 mL of AIM-V medium, 4.45 mL of HFDM-1(+), and 0.5 mL of NeoSERA.

[0226] When the density of cells cultured in a 90 mm dish reaches 70-95%, P1 cells are collected and transferred to a cell culture flask (#MS-23800S 225 cm 2 :Sumitomo Bakelite Co., Ltd.) were inoculated with 2.5×10 P1 cells 5 The culture medium for one cell culture flask is 25 mL of AIM-V medium, 25 mL of HFDM-1(+), and 1 mL of autologous serum.

[0227] The culture medium was removed from the cell culture flask by aspiration, and 25 mL of PBS (Cell Science & Technology Institute, Inc.) was added to each cell culture flask to wash the cell culture flask. The PBS was then removed by aspiration.

[0228] · Add 7 mL of recombinant trypsin / EDTA solution (r-TE (Cell Science & Technology Institute, Inc.)) to a cell culture flask and place the cell culture flask in an incubator (37°C, 5% CO 2 ) and let it stand for 3 minutes.

[0229] A 40 μm cell strainer (#352340: Corning Incorporated) was installed in a 50 mL tube.

[0230] The cells were detached from the cell culture flask by pipetting and transferred to the above 50 mL tube.

[0231] · 7 mL of s-TI (Cell Science & Technology Institute, Inc.), a synthetic trypsin neutralizer solution, was added to one cell culture flask, and the inside of a 10 cm culture dish was washed, and the washing solution was transferred to the above-mentioned 50 mL tube.

[0232] Centrifuge the 50 mL tube at 1200 rpm for 2 minutes at 20°C.

[0233] After centrifugation, the supernatant was removed by aspiration and the cells were suspended in 10 mL of HFDM-1(+) (Cell Science & Technology Institute, Inc.) to obtain a suspension containing P2 cells. 10 μL of this cell suspension was mixed with 10 μL of trypan blue and smeared on a hemocytometer to count the cells.

[0234] A portion of P2 cells was adjusted to 1×10 using STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: ZENIAQ Resource Co., LTD.). 6 Cells were stored at -80°C until use.

[0235] One of the above-mentioned cryovials (containing the cell preservation solution of P2 cells) that had been frozen and stored at -80°C for a predetermined period of time was thawed, and the cell survival rate was measured by the following method.

[0236] 8.5 mL of AIM-V medium, 0.5 mL of NeoSERA, and the thawed preservation solution for the above-mentioned P2 cells were added to a 15 mL tube, and the tube was centrifuged (1200 rpm, 2 min, 20°C).

[0237] The supernatant was removed by aspiration, and the cells were suspended in 2 mL of AIM-V medium. 10 μL of the suspension was used to count the number of cells using the same method as in Example 1, and the cell survival rate of P2 was measured.

[0238] Seed cells onto two 10 cm culture dishes.

[0239] 10 cm culture dish: AIM-V medium 5 mL + HFDM-1 (+) (Cell Science & Technology Institute, Inc.) + NeoSERA 1 mL, cell suspension 1 mL

[0240] After culturing for 4 to 10 days, cells were detached from a 10 cm dish with r-TE / s-TI and centrifuged. The cells were then suspended in 2 mL of AIM-V medium and 10 μL of the suspension was used to count the cells and determine the cell viability.

[0241] The results are shown in Table 6. According to Table 6, even after storage at -80°C for 434 to 1457 days, the cell viability was maintained at a high level of 71 to 88.5%.

[0242] [Table 6]

[0243]

[0244] [Example 5] Preparation of cell sheets

[0245] Cell sheets were prepared using mesenchymal cells (No. 1 to No. 5) by the following method.

[0246] In Example 1, for the doubling experiment, three cell culture flasks (#MS-23800 225 cm 2 :Sumitomo Bakelite Co., Ltd.) were inoculated with 2.5×10 P3 cells 5 Cells were passaged 3 days after seeding for No. 1 and 5 days after seeding for No. 2 to 5. During the passage, cells were removed from the cell culture flask and the number of cells was counted with a cell counter. For passage, cells P4 were seeded into the cell culture flask and 5×10 mesenchymal cells were seeded into one well of a 24-well plate. 5 Cells were cultured for 72 hours, and the supernatant was stored for ELISA analysis. Cell sheets were prepared from P4 cells using the method described in Patent Document 3. These sheets were attached to the CellShifter™ to prevent shrinkage, fixed with 10% neutral formalin, and sectioned.

[0247] The sections of the cell sheets prepared by conventional methods were stained with hematoxylin and eosin (HE) and MT, and the photographs (4 times) are shown in FIG. Figure 1.pass Figure 1 HE staining confirmed that the cells formed a cell sheet structure. Furthermore, MT staining confirmed that the cell sheet contained a rich extracellular matrix. Cell sheets were also produced using P3 cells and P5-P10 cells (not shown).

[0248] [Example 6] Expression of cell surface antigen molecules in cells collected from oral tissues

[0249] Although cells collected from gums were used in Example 2 above, analysis of cell surface antigen molecules was performed in the same manner as in Example 2 using P4 cells (4 samples) collected from the lower half of the salivary gland (the inner side of the cheek) tissue. The cells and culture methods used were the same as in Example 4, and the analysis methods of cell surface antigen molecules and the antibodies or reagents used were the same as in Example 2. The results are shown in Table 7. It was confirmed that 99% of the cells expressing CD90, CD73, CD44, CD13, CD29 or CD59 were included in the cell population obtained by collecting the mesenchymal cells from the lower half of the salivary gland (the inner side of the cheek) tissue, and that CD105 included 27% to 35%, CD166 included more than 62%, CD49e included more than 56%, CD151 included more than 93%, CD280 included more than 97%, and CD63 included more than 63%. On the other hand, cells expressing CD34 and CD45 were not confirmed.

[0250] [Table 7]

[0251]

[0252] [Example 7] Analysis of cell surface antigens in a cell population obtained by culturing mesenchymal cells collected from gingiva and treated with IFN-γ

[0253] The cell surface antigen analysis (HLA class I, HLA class II, CD106, CD274) of the cell population obtained by culturing the IFN-γ-treated mesenchymal cells was performed by the following procedure.

[0254] One cryovial prepared in Example 1 (containing the P3 cell preservation solution) was taken out from -80°C and thawed.

[0255] · 3.5 mL of AIM-V medium, 0.5 mL of NeoSERA, and thawed cell storage medium were added to a 15 mL tube, and the tube was centrifuged (1200 rpm, 1 min, 20°C).

[0256] Remove the supernatant by aspiration, inoculate two 10 cm culture dishes, and culture for 3 days (37°C, 5% CO2). For each 10 cm culture dish, use 5 mL of AIM-V medium, 5 mL of HFDM-1(+), and 1 mL of NeoSERA.

[0257] The cells were washed with PBS and then detached with r-TE / s-TI. The cells were centrifuged (1200 rpm, 1 min, 20°C). The supernatant was removed by aspiration and the cells were suspended in 2 mL of AIM-V medium. 10 μL of the suspension was used for cell counting.

[0258] · Each cell was seeded into two 10 cm culture dishes and cultured overnight. 3×10 5 cells of cells.

[0259] 1 10cm culture dish: 5.5mL AIM-V medium + 4.5mL HFDM-1(+), 1mL NeoSERA

[0260] To ten 15 mL tubes, add 4.5 mL of AIM-V medium, 4.5 mL of HFDM-1(+), and 1 mL of NeoSERA.

[0261] To each of the five 15 mL tubes, add 5 μL of 0.2 mg / mL (R&D Recombinat Human IFN-r#285-IF / CF, dissolved in Otsuka distilled water (Otsuka Pharmaceutical Factory, Inc.)) (final concentration: 100 ng / mL).

[0262] To each of the other five 15 mL tubes, 5 μL of Otsuka distilled water (Otsuka Pharmaceutical Factory, Inc.) was added.

[0263] The culture medium was removed by aspiration from the 10 cm culture dish, and the above-mentioned adjusted medium was added. The cells were cultured for 48 hours, and then analyzed for cell surface antigens.

[0264] Cell surface antigen analysis was performed using the same method as in Example 2. Antibodies listed in Table 3 were used. The results are shown in Tables 8 and 9. Furthermore, since CD106 and C274 were independently tested, they are shown separately in the table.

[0265] Table 8 confirms that IFN-γ treatment increases HLA class II expression. Furthermore, no expression of CD106 was observed, regardless of IFN-γ treatment. Furthermore, Table 9 confirms that IFN-γ treatment increases not only HLA class II expression but also CD274 expression, with a positive rate of 91-96%.

[0266] [Table 8]

[0267]

[0268] [Table 9]

[0269]

[0270] Although the immunogenicity of MSCs, which are mesenchymal stem cells, is low, it has been reported that they acquire immunogenicity through HLA class II (MHC class II) expression in an inflammatory environment. In addition, it has been reported that cells expressing CD274 (PD-L1) have the effect of suppressing the function of surrounding immune cells (Yu Yoshinaga et al., Stem Cell Reports. 2022; 17: 1714-1729.). Therefore, it is believed that in cell transplantation therapy based on allogeneic cells, if the transplanted allogeneic cells express CD274, the immunogenicity of the transplanted allogeneic cells will be reduced by suppressing the attack of immune cells from the organism. The proportion of cells expressing CD274 in the cell population cultured in the present invention is more than 60%, but by culturing in a culture medium supplemented with IFN-γ, the proportion of cells expressing CD274 in the cell population reaches more than 91%. This result is considered to indicate that when a cell population obtained by culturing mesenchymal cells cultured according to the present invention is transplanted as allogeneic cells, a strong immune rejection reaction can be reduced by suppressing the attack of immune cells from the organism.

[0271] [Example 8] VEGF secretion

[0272] For use as a cell sheet, VEGF secretion is crucial. Therefore, VEGF secretion from the cell sheet was examined. The supernatant from the cell sheet prepared from mesenchymal cells collected from gingival tissue, which was used for ELISA analysis during the preparation of the cell sheet in Example 5, was used. The supernatant from the cell sheet prepared from mesenchymal cells collected from the lower half of the salivary gland (inner cheek) tissue was prepared using the following method.

[0273] The P2 cell preservation solution prepared from mesenchymal cells collected from the lower half of the salivary gland (inner cheek) prepared in Example 4 was taken out from -80°C and thawed. P3 cells were prepared from P2 cells in the same manner as described in Example 1 and plated in a 24-well plate (1.8 cm 2 / well, MS-80240: Sumitomo Bakelite Co., Ltd.) inoculated with 2.78×10 5 pieces / cm 2 (The seeding number in one well is 5.0×10 5 The cells were cultured in an incubator (37°C, 5% CO2) for 72 hours, and the culture solution was transferred to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes at 4°C. The supernatant was used for ELISA analysis and stored at -80°C.

[0274] For ELISA analysis, on the day of the assay, 1.5 mL tubes containing the supernatants for each ELISA assay were thawed on ice. The VEGF concentration in the supernatant was then measured using the Human VEGF Quantikine ELISA Kit (R&D Systems, #DVE00). The absorbance was measured and the concentration was calculated using the iMark Microplate Reader (BIO-RAD) and MPM6.exe (BIO-RAD).

[0275] (result)

[0276] The VEGF concentration in the culture medium when mesenchymal cells collected from the lower half of the salivary gland (inner cheek) and mesenchymal cells collected from the gingival tissue were cultured to prepare cell sheets is shown in FIG. Figure 2 .Depend on Figure 2 It was found that all cell sheets secreted sufficient VEGF, but it was confirmed that the cell sheet prepared using mesenchymal cells collected from gingival tissue secreted at least twice as much VEGF as the cell sheet prepared using mesenchymal cells collected from the lower half of the salivary gland (inner cheek) tissue.

[0277] [Example 9] Regarding the presence or absence of SSEA-3 expression

[0278] SSEA-3 is a cell surface antigen molecule known as a representative marker of pluripotent stem cells. Whether gingival mesenchymal cells express SSEA-3 was investigated.

[0279] Using one cryovial (containing a cell preservation solution of P3 cells) prepared from gingival mesenchymal cells from five donors No. 1 to 5 using the method described in Example 1, SSEA-3 expression was examined using the same method as in Example 2. Antibodies used were anti-SSEA-3 antibody (#330311: PE anti-human / mouse SSEA-3 Antibody, BioLegends) and a control (#400807: PE Rat IgM, κ Isotype Ctrl Antibody, BioLegends). The results are shown in Figure 3 .

[0280] Depend on Figure 3 It can be seen that the peak did not shift to the right and the positive rate value did not change. Therefore, it was confirmed that gingival mesenchymal cells did not express SSEA-3.

Claims

A cell population isolated from a living organism, comprising 80% or more of mesenchymal cells expressing at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

2. The cell population according to claim 1, wherein Mesenchymal cells are cells collected from oral tissues or their cultured form.

3. The cell population according to claim 1 or 2, characterized in that Mesenchymal cells express at least three or more cell surface antigen molecules selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. The cell population according to claim 1 or 2, comprising 60% or more of mesenchymal cells expressing CD274. The cell population according to claim 1 or 2, comprising less than 5% of mesenchymal cells expressing SSEA-3. A three-dimensional cell culture comprising the cell population according to claim 1 .

7. A pharmaceutical composition comprising the cell population according to claim 1 or 2. The pharmaceutical composition according to claim 7 , further comprising a pharmaceutically acceptable additive. 9 . The pharmaceutical composition according to claim 7 , which is in the form of a three-dimensional cell culture. 10 . The pharmaceutical composition according to claim 7 or 8 , which is in the form of a cell sheet.

11. Exosomes obtained from the cell population according to claim 1 or 2.

12. A method for producing the cell population according to claim 1 or 2, comprising: (a) a step of collecting mesenchymal cells from living tissue, and (b) A step of culturing the collected mesenchymal cells.

13. The method according to claim 12, further comprising after step (b): (c) a step of confirming the expression of the cell surface antigen molecule using an antibody that recognizes at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

14. The method according to claim 12 or 13, characterized in that In step (b), the cells are cultured in a medium containing inflammatory cytokines. 15 . The method according to claim 12 or 13 , further comprising, after step (b), the step of: (d) cryopreserving the cultured cells.

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