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

Abstract

The present disclosure addresses the problem of providing a cell population isolated from a living body and comprising mesenchymal cells that have a prescribed cell surface antigen molecule and can be used to produce a cell sheet.　The present invention uses a cell population isolated from a living body and comprising at least 80% mesenchymal cells that express at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. It is preferable that the mesenchymal cells are cells collected from intraoral tissue or are a culture thereof, and that the mesenchymal cells express at least three cell surface antigen molecules selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

Description

Cell population containing mesenchymal cells, pharmaceutical composition containing the cell population, exosomes obtained from the cell population, and method for producing the cell population

[0001] The present disclosure relates to a cell population containing mesenchymal cells that express a specific cell surface antigen molecule, a three-dimensional cell culture containing the cell population, a pharmaceutical composition containing the cell population, exosomes obtained from the cell population, and a method for producing the cell population.

[0002] In recent years, cell transplantation therapy has attracted attention as an effective treatment for various diseases and tissue damage. When simply transplanting cells, they have difficulty implanting into tissue. Therefore, cell transplantation therapy uses cell sheets, in which the cells to be transplanted are cultured in a sheet form. Using this cell sheet makes it possible for the transplanted cells to settle in the desired tissue.

[0003] The present inventors have been developing cell sheets made of cultured fibroblasts, and have disclosed a method for producing a cell sheet that includes a step of culturing the cells for a predetermined period of time under a predetermined temperature and low-oxygen conditions (see Patent Document 1), a cell sheet containing peripheral blood mononuclear cells and fibroblasts that is used as a transplant material for treating intractable skin ulcers (see Patent Document 2), and a method for producing a layered cell sheet that includes a step of seeding and culturing predetermined fibroblasts on a culture substrate to which a medium has been added (see Patent Document 3).

[0004] Fibroblasts are used in each of the above cell sheets. Fibroblasts are one of the cells that make up connective tissue, and are thought to be cells that exist in the dermis and produce dermal components such as collagen, elastin, and hyaluronic acid. Fibroblasts are found throughout the body and are characterized by their strong proliferation ability. However, little progress has been made in research into the characteristics of cell surface antigen molecules. Fibroblasts expressing VCAM-1 (vascular cell adhesion molecule 1: CD106) have been disclosed as cardiac cell culture materials (see Patent Document 4).

[0005] Fibroblasts are cells of mesenchymal origin. To date, mesenchymal stem cells that highly express at least one cell surface marker selected from the group consisting of EGFR and MIC-AB (see Patent Document 5) and mesenchymal stem cells 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) have been disclosed.

[0006] Furthermore, with regard to stem cells derived from the oral cavity or gingiva, it has been disclosed that the CD56 positivity rate in a cell population of fibroblasts derived from oral mucosa is 43% (see Non-Patent Document 1), that gingiva-derived stem cells express existing MSC cell surface markers, and therefore the CD105 positivity rate is 99% (see Non-Patent Document 2), that gingiva-derived stem cells are positive for CD29, CD44, CD90, and CD105, but negative for CD45 and CD106 (see Non-Patent Document 3), and that a review on gingiva-derived mesenchymal stem cells and progenitor cells discloses the expression of cell surface antigen molecules (see Non-Patent Document 4), and that a cell population of gingiva-derived mesenchymal stem cells has a CD105 positivity rate of 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. Generally, it takes 25 to 53 hours for cells to double in number (doubling time) (see Non-Patent Document 6).

[0008] International Publication No. 2016 / 043201 Pamphlet International Publication No. 2016 / 068217 Pamphlet Japanese Patent Application Laid-Open No. 2019-38 Japanese Patent Application Laid-Open No. 2016-27797 International Publication No. 2017 / 170925 Pamphlet International Publication No. 2017 / 188403 Pamphlet Japanese Patent Application Laid-Open No. 2022-120128

[0009] Higa, K. et al. Future Sci. OA (2017) 3(4), FSO243Lingqian Du. et al. Journal of Dental Sciences (2016) 11, 304e314Ihsene Taihi et al. Stem Cell Research & Therapy (2022) 13:125Karim M. Fawzy El-Sayed et al. Stem Cells International Volume 2016, Article ID 7154327, 16 pages.Tomas I Mitrano et al. J Periodontol. 2010 Jun;81(6):917-25.Endo Tomohiko et al., Journal of Dental Health (1995) 45,322-333

[0010] An object of the present disclosure is to provide a cell population isolated from a living organism, which contains mesenchymal cells that have a specific cell surface antigen molecule and can be used to prepare a cell sheet.

[0011] The present inventors have been conducting research into allogeneic (allogeneic) cell sheet transplantation for the prevention of postoperative complications and the treatment of intractable skin ulcers. During this research, they focused on the gingiva, which is removed and discarded when young people in their teens and twenties have their wisdom teeth removed. They then analyzed the characteristics of cell surface antigen molecules on mesenchymal cells collected from oral tissues, leading to the completion of this disclosure.

[0012] That is, the present disclosure is as follows: [1] A cell population isolated from a living body, comprising 80% or more 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 [1] above, characterized in that the mesenchymal cells are cells collected from oral tissue or a culture thereof. [3] The cell population according to [1] or [2] above, characterized in that 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. [4] The cell population according to [1] or [2] above, comprising 60% or more mesenchymal cells expressing CD274. [5] The cell population according to [1] or [2] above, comprising 5% or less mesenchymal cells expressing SSEA-3. [6] A three-dimensional cell culture comprising the cell population described in [1] or [2] above. [7] A pharmaceutical composition comprising the cell population described in [1] or [2] above. [8] The pharmaceutical composition described in [7] above, further comprising a pharmaceutically acceptable additive. [9] The pharmaceutical composition described in [7] or [8] above, in the form of a three-dimensional cell culture.

[10] The pharmaceutical composition described in [7] or [8] above, in the form of a cell sheet.

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

[12] A method for producing the cell population described in [1] or [2] above, comprising the following steps in sequence: (a) collecting mesenchymal cells from a biological tissue; and (b) culturing the collected mesenchymal cells.

[13] The method of

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

[14] The method of

[12] or

[13] above, characterized in that in step (b), the cells are cultured in a medium containing an inflammatory cytokine.

[15] The method of

[12] or

[13] above, further comprising, after step (b), (d) a step of cryopreserving the cultured cells.

[0013] Further, other aspect 1 of the present disclosure is as follows. [1'] A cell population isolated from a living organism, comprising 80% or more 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 [1'] above, characterized in that the mesenchymal cells are cells collected from oral tissue or a culture thereof. [3'] The cell population according to [1'] or [2'] above, characterized in that 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. [4'] The cell population according to any of [1'] to [3'] above, comprising 60% or more mesenchymal cells expressing CD274. [5'] The cell population according to any of [1'] to [4'] above, comprising 5% or less mesenchymal cells expressing SSEA-3. [6'] A three-dimensional cell culture comprising the cell population described in any of [1'] to [5'] above. [7'] A pharmaceutical composition comprising the cell population described in any of [1'] to [5'] above. [8'] The pharmaceutical composition described in [7'] above, further comprising a pharmaceutically acceptable additive. [9'] The pharmaceutical composition described in [7'] or [8'] above, in the form of a three-dimensional cell culture. [10'] The pharmaceutical composition described in any of [7'] to [9'] above, in the form of a cell sheet. [11'] Exosomes obtained from the cell population described in any of [1'] to [5'] above. [12'] A method for producing the cell population described in any of [1'] to [5'] above, comprising the steps of: (a) collecting mesenchymal cells from a biological tissue; and (b) culturing the collected mesenchymal cells. [13'] The method of [12'] above, further comprising, after step (b), (c) a step of confirming the expression of at least one cell surface antigen molecule using an antibody that recognizes the cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. [14'] The method of [12'] or [13'] above, characterized in that in step (b), the cells are cultured in a medium containing an inflammatory cytokine. [15'] The method of any of [12'] to [14'] above, further comprising, after step (b), (d) a step of cryopreserving the cultured cells.

[0014] Further, another aspect 2 of the present disclosure is as follows:

[16] A method for preventing or treating ischemic tissue, intractable skin ulcer, burn, ischemic heart disease, critical limb ischemia, or postoperative complication, characterized by transplanting the pharmaceutical composition according to any of [7'] to [10'] above into a subject in need of transplantation.

[17] Use of the cell population according to any of [1'] to [5'] above for producing a pharmaceutical composition for preventing or treating ischemic tissue, intractable skin ulcer, burn, ischemic heart disease, critical limb ischemia, or postoperative complication.

[0015] The cell population of the present disclosure enables cell transplantation therapy and the production of cell sheets for use in such therapy. Furthermore, if mesenchymal cells collected from the gums are cultured to produce a cell population, they can be easily collected from the gums of healthy individuals at the time of dental treatment, etc. Furthermore, the cell population of the present disclosure rapidly proliferates, enabling the rapid production of cell sheets.

[0016] Fig. 1 is a photograph of a cell sheet prepared in Example 5. Fig. 2 is a diagram showing the results of examining the secretion of vascular endothelial growth factor (VEGF) from a cell sheet in Example 8. Fig. 3 is a diagram showing the results of examining the expression of SSEA-3 in gingival-derived mesenchymal cells in Example 9.

[0017] <Cell Population> The cell population of the present disclosure is not particularly limited as long as it is a cell population isolated from a living organism, which contains 80% or more mesenchymal cells that express at least one cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280, and hereinafter may be referred to as the "cell population of the present invention."

[0018] As used herein, "mesenchymal cells" refers to a general term for cells present in mesenchymal tissue, which is a connective tissue formed between the ectoderm and endoderm during development, and an example thereof is fibroblasts. Furthermore, preferred examples of mesenchymal cells include mesenchymal cells collected from oral tissues such as the gums or the lower part of the salivary glands (inside the cheek), adipose tissue, bone marrow, or skin, or cultures thereof. The term "cultures thereof" refers to cultured mesenchymal cells in which the expression of cell surface molecules in the mesenchymal cells is maintained.

[0019] As used herein, "fibroblasts" refer to a cell type that contributes to the formation of connective tissue, a fibrous material that maintains and connects tissues and organs in the body. While they have no particularly significant functions in normal tissues, upon injury, they migrate to the wound site and secrete collagen, elastin, hyaluronic acid, and the like, which help maintain the tissue network, thereby initiating the production of extracellular matrix and renewing the extracellular matrix. Additionally, they play an important role in the wound healing process, such as inducing wound contraction. Methods for preparing fibroblasts include the method described in Patent Document 2 above, as well as the method for preparing mesenchymal cells described below, but are not limited thereto. Any cell population obtained under conditions commonly used in the art for preparing fibroblast fractions may be used.

[0020] The origin of the 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. The term "origin" refers to the animal species from which the cells are collected.

[0021] The cluster of differentiation (CD) classification of cell surface antigen molecules herein is based on the CD classification listed on the HGNC website (www.genenames.org / data / genegroup / #! / group / 471). Gene names herein are based on the gene names listed on the GeneCards (registered trademark) website (www.genecards.org).

[0022] The present cell population preferably contains 80% or more of the above mesenchymal cells, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. Here, the mesenchymal cells contained in the present cell population may be one type or a combination of two or more types, so 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 present cell population may contain, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more mesenchymal cells that express 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 mesenchymal cells that express two, three, four, or five types selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280.

[0023] The mesenchymal cells used 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, it is preferable for them to express at least two types selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280, it is more preferable for them to express at least three types selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280, it is even more preferable for them to express at least four types selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280, and it is particularly preferable for them to express CD13, CD59, CD49e, CD151, and CD280.

[0024] When at least two types selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least two types may be any of 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; or CD151 and CD280.

[0025] When at least three types selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least three types may be any of 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.

[0026] When at least four types selected from the group consisting of CD13, CD59, CD49e, CD151 and CD280 are expressed, the at least four types may be any of CD13, CD59, CD49e and CD151; CD13, CD59, CD49e and CD280; CD13, CD59, CD151 and CD280; CD13, CD49e, CD151 and CD280; and CD59, CD49e, CD151 and CD280.

[0027] The above-mentioned cell population can further include a cell population containing mesenchymal cells expressing CD29, CD63, CD90, CD73, CD44, CD13, and / or HLA class I in an amount of 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0028] Furthermore, the above-mentioned cell population can include a cell population containing mesenchymal cells expressing CD304, CD106, CD34, and / or CD45 in an amount of 10% or less, preferably 5% or less, more preferably 3% or less, and even more preferably 1% or less.

[0029] Furthermore, the cell population may include a cell population containing 80% or less, preferably 70% or less, and more preferably 15 to 68% of mesenchymal cells expressing CD105.

[0030] Additionally, the 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% mesenchymal cells that express SSEA-3 (Stage-Specific Embryonic Antigen-3), which is known as a cell surface antigen molecule that is a marker for pluripotent stem cells.

[0031] When the pharmaceutical composition contains the above-mentioned cell population and is in the form of a cell sheet to be used in allogeneic transplantation, the cell population may contain mesenchymal cells that express CD274 in an amount of 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, in order to suppress immune rejection.

[0032] The expression of the cell surface antigen molecule on the cells can be confirmed, for example, using an antibody capable of binding to the cell surface antigen molecule. Specifically, in the case of confirming the expression of CD13, a suspension of the cell population can be prepared, and the expression of CD13 on the cells constituting the cell population can be confirmed using an anti-CD13 antibody.

[0033] As used herein, "express" means that the protein of the cell surface antigen molecule is expressed, and it is preferable that the cell surface antigen molecule is presented on the cell surface.

[0034] Furthermore, in the present cell population, cells other than the above-mentioned mesenchymal cells are not particularly limited, but examples thereof include lymphocytic 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.

[0035] <Three-dimensional cell culture construct> The three-dimensional cell culture construct herein is not particularly limited as long as it contains the cell population of the present invention. The form of the three-dimensional cell culture construct is one in which cells are physically and functionally connected to each other via adhesion molecules, extracellular matrix, etc., to form the desired three-dimensional structure. Here, "three-dimensional cell culture construct" includes, but is not limited to, cell sheets, spheroids, organoids, etc. The shape of the three-dimensional cell culture construct includes, but is not limited to, sheet, organ, spherical, tissue, hollow, block, etc. Typically, as in cell sheets, adherent cells (cells that adhere to and grow on a culture substrate, e.g., fibroblasts) tend to form three-dimensional cell aggregates through physical and / or functional connections between cells. However, even suspension cells (i.e., cells that grow suspended in a medium, e.g., hematopoietic cells, blood cells), such as spheroids, which form three-dimensional cell aggregates through physical and / or functional connections between cells, are considered to be three-dimensional cell culture constructs herein.

[0036] The method for producing a three-dimensional cell culture construct may be any method known to those skilled in the art and is not particularly limited. Examples of the method for producing a three-dimensional cell culture construct include methods taught in documents such as JP 2012-120696 A, JP 2017-176025 A, and JP 2015-149905 A.

[0037] The thickness of the three-dimensional cell culture construct 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.

[0038] In a three-dimensional culture containing a cell population, the cell density of the three-dimensional culture to be cultured is, for example, 5 × 10 １ cells / cm ３ 5x10 or more １０ cells / cm ３ More preferably, it is 5×10 ２ cells / cm ３ 5x10 or more ９ cells / cm ３ or less, more preferably 5 × 10 ３ cells / cm ３ 1x10 or more ９ cells / cm ３ Below, particularly preferably 5 × 10 ４ cells / cm ３ 5x10 or more ８ cells / cm ３ The following is the result.

[0039] <Pharmaceutical Composition> The pharmaceutical composition of the present disclosure is not particularly limited as long as it contains the cell population, and hereinafter may be referred to as the "pharmaceutical composition of the present invention." The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable additive. The pharmaceutical composition of the present invention may be in the form of a suspension, a three-dimensional cell culture construct, or the like, and is preferably in the form of a cell sheet.

[0040] The present pharmaceutical composition is preferably a pharmaceutical composition for ischemic tissue treatment. Here, "ischemic tissue" refers to tissue in a state of reduced blood flow. In ischemic tissue, tissue breakdown occurs as blood flow decreases. The present pharmaceutical composition promotes angiogenesis in such ischemic tissue through the secretion of VEGF, thereby improving blood flow. Diseases to be treated include intractable skin ulcers, such as those caused by pressure ulcers, arteriosclerosis obliterans, diabetes, venous insufficiency, collagen disease, and vasculitis. Furthermore, when using the present pharmaceutical composition to prevent or treat intractable skin ulcers, the present pharmaceutical composition can be transplanted into a subject requiring transplantation, such as a patient with an intractable skin ulcer. Other diseases to be treated include ischemic tissue, burns, ischemic heart disease, critical limb ischemia, and postoperative complications (e.g., anastomotic failure of various organs, bronchial stump fistula, pancreatic fistula, and bile leakage).

[0041] Furthermore, the pharmaceutical composition of the present invention can be used to treat intractable skin ulcers such as ischemic ulcers, ulcers associated with collagen diseases, and ulcers associated with radiation therapy, to prevent postoperative complications, and as a supplementary agent for the purpose of such prevention.

[0042] As used herein, "pharmaceutically acceptable excipients" include saline, buffered saline, cell culture media, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonicity agents, bulking agents, and lubricants.

[0043] The cell sheet form in the present pharmaceutical composition is a form in which cells are physically and functionally connected to each other directly or via adhesion molecules, extracellular matrix, etc. to form a sheet structure. The method for producing a cell sheet may be performed using methods known to those skilled in the art and is not particularly limited. Examples of methods for producing a cell sheet include methods taught in literature such as Patent Documents 1 to 3, JP 2011-006490 A, WO 2015 / 068505 A, and JP 2022-8269 A.

[0044] 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, but examples include multi-layer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0045] The thickness of the cell sheet used herein is not particularly limited. The thickness of the cell sheet is, for example, 0.001 mm or more and 2.0 mm or less. More preferably, it is 0.01 mm or more and 1.5 mm or less, even more preferably, 0.03 mm or more and 1.2 mm or less, and particularly preferably, it is 0.05 mm or more and 1.0 mm or less. By setting the thickness of the cell sheet within the above range, a high cell survival rate within the cell sheet and excellent shape-retention ability that is advantageous for cell transplantation can be exhibited.

[0046] The term "suspension form" as used herein refers to a state in which cells are suspended in a solution such as a medium, PBS, or physiological saline, either alone or in contact with other cells.

[0047] <Exosomes> The exosomes herein are not particularly limited as long as they are obtained from the present cell population, and are hereinafter also referred to as "the present exosomes." As used herein, "exosomes" refers to cell-derived vesicles composed of a membranous bilayer. Exosomes are released from most cell types and can be found in many body fluids. They encapsulate nucleic acids such as DNA, mRNA, or microRNA, as well as proteins. In a preferred embodiment, the exosomes herein are obtained from the present cell population containing mesenchymal cells collected from human gingiva. Exosomes obtained from the present cell population are preferred for their anti-inflammatory and wound healing effects, their ease of collection due to the ability to culture mesenchymal cells without serum, their low risk of carcinogenesis, and their high safety. Furthermore, they encapsulate various growth factors. The size of the present exosomes can range, for example, from 20 nm to 140 nm in diameter.

[0048] The present exosome production method involves culturing the present cell population, subjecting the culture supernatant to centrifugation or ultrafiltration to remove cells and impurities, and then recovering the supernatant. Centrifugation can be performed, for example, at 1,000 to 3,000 rpm for 2 to 10 minutes. Further purified exosomes can then be recovered using a commercially available exosome isolation kit, or exosomes can be purified and recovered by ultracentrifugation (e.g., 100,000 g to 1,000,000 g, 30 minutes to 12 hours).

[0049] <Method for producing a cell population> The method for producing a cell population in the present specification is not particularly limited as long as it includes, in order, (a) the step of collecting mesenchymal cells from biological tissue; and (b) the step of culturing the collected mesenchymal cells, and hereinafter this method will also be simply referred to as the "method for producing the present cell population."

[0050] After step (b), the method may include, but is not limited to, (c) a step of confirming the expression of at least one cell surface antigen molecule using an antibody that recognizes the cell surface antigen molecule selected from the group consisting of CD13, CD59, CD49e, CD151, and CD280. Furthermore, after step (b), the method may include, but is not limited to, (d) a step of cryopreserving the cultured cells. The cryopreservation period and cryopreservation method will be described later.

[0051] The method for culturing cells is not particularly limited, and can be a conventional method used in the fields of regenerative medicine, pharmaceuticals, quasi-drugs, etc. Specific examples include a method in which a culture medium is added to a culture vessel, cells to be cultured are seeded, and the cells are cultured under an optimal environment until the desired state is reached.

[0052] The culture medium used in the above-mentioned cell culture method is a solution containing components necessary for cell culture. The culture medium is not particularly limited as long as it is suitable for the cells to be cultured. Examples of culture medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, additives, etc.

[0053] These culture medium components may be blended alone or in combination of two or more. These culture medium components can be appropriately prepared from known components depending on the cells to be cultured, etc.

[0054] Examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0055] Examples of 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.

[0056] Examples of vitamins include sodium ascorbate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0057] Examples of inorganic salts include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate.

[0058] Examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0059] Examples of additives include serum such as fetal bovine serum, horse serum, and human serum; growth factors such as FGF2, EGF, HGF, VEGF, and PDGF; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin and streptomycin; pH adjusters such as HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid); organic acids such as lactic acid and propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine and putrescine; reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan; and Rho kinase inhibitors.

[0060] When the cells to be cultured are stem cells or progenitor cells, differentiation induction treatment may be performed during culture by adding a differentiation-inducing factor to the culture medium, such as activin A, BMP4, bFGF, VEGF, SCF, DKK1, BMP signal inhibitor, TGFβ / activin / NODAL signal inhibitor, Wnt signal inhibitor, and retinoic acid signal inhibitor.

[0061] In the step (b), the cells may be cultured in a 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.

[0062] 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. Alternatively, the expression of each cell surface molecule may be confirmed using an antibody that recognizes CD13, CD59, CD49e, CD151, or CD280. Furthermore, the percentage (%) of cells expressing each cell surface molecule in the cell population can be determined by counting the number of cells constituting the cell population and the number of cells expressing each cell surface molecule. Whether or not the cells are mesenchymal cells can be confirmed using an antibody that recognizes CD90.

[0063] Specific examples of culture media containing the above-mentioned culture media components include AIM (registered trademark) V medium, HFDM-1 Medium, equilibrated buffers such as Dulbecco's phosphate buffered saline (D-PBS) and 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), and 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 Examples of suitable culture media include medium, Williams' medium E, Brinster's BMOC-3 medium, and E8 medium.

[0064] The culture conditions are not particularly limited as long as they can bring the cultured cells into the desired state. Typical culture conditions include, for example, culture at 37°C, 5% CO using a regulated basal culture medium. ２ The culture conditions may be appropriately set depending on the cells to be cultured, etc. The cell culture period is not particularly limited as long as the cultured cells reach the desired state. The cell culture period is, for example, within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, or within 3 days.

[0065] The number of cells seeded in the culture vessel is not particularly limited as long as it is suitable for the cells to be cultured and the culture vessel. For example, the number of cells seeded in the culture vessel is 1 × 10 ３ Cells / mL or more 1 x 10 ７ cells / mL or less, more preferably 1 x 10 ４ Cells / mL or more 1 x 10 ６ cells / mL or less, more preferably 5 x 10 ４ Cells / mL or more 5 x 10 ６ cells / mL or less, particularly preferably 1 x 10 ５ Cells / mL or more 1 x 10 ６ cells / mL or less.

[0066] The density of the cells to be cultured is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel, and the intended use of the cultured cells. The planar density of the cells to be cultured is, for example, 1 × 10 ４ cells / cm ２ 1x10 or more ９ cells / cm ２ More preferably, it is 1×10 ５ cells / cm ２ 1x10 or more ８ cells / cm ２ More preferably, 2 × 10 ５ cells / cm ２ 1x10 or more ７ cells / cm ２ Below, particularly preferably 5 × 10 ５ cells / cm ２ 1x10 or more ６ cells / cm２ The following is the result.

[0067] When a pharmaceutical composition containing a cell population is prepared as a cell sheet or three-dimensional culture containing the cell population, the cell density of the cell sheet or three-dimensional culture to be cultured is, for example, 5 × 10 １ cells / cm ３ 5x10 or more １０ cells / cm ３ More preferably, it is 5×10 ２ cells / cm ３ 5x10 or more ９ cells / cm ３ or less, more preferably 5 × 10 ３ cells / cm ３ 1x10 or more ９ cells / cm ３ Below, particularly preferably 5 × 10 ４ cells / cm ３ 5x10 or more ８ cells / cm ３ The following is the result.

[0068] The mesenchymal cells herein may be autologous cells derived from the patient for whom the pharmaceutical composition is to be used, or allogeneic cells derived from another subject of the same species, for example, another patient.

[0069] The dosage of the pharmaceutical composition of the present invention is 5 x 10 cells in suspension. ２ ~1 x 10 １２ pieces / time, preferably 1 x 10 ４ ~1 x 10 １１ pieces / time, more preferably 1 x 10 ５ ~1 x 10 １０ This dose may be administered multiple times as a single dose, or may be administered in divided doses. Generally, when administered to an adult, the number of cells per body weight is 1 x 10 to 5 x 10 １０ pieces / kg, preferably 1 x 10 ２ ~5 x 10 ９ pieces / kg, more preferably 1 x 10 ３ ~5 x 10 ８The cell density is 1 / kg. This dose may be administered multiple times as a single dose, or may be administered in multiple divided doses. <Cryopreservation> The cell population, the three-dimensional cell culture construct, the pharmaceutical composition, and the exosomes may be cryopreserved. Specifically, they can be cryopreserved at -20°C or below, preferably -80°C, for one day or more, one week to ten years, one month to five years, or one year to three years. Examples of freezing methods include freezing using a cell cryopreservation solution widely used in bioengineering, the freezing method described in JP 2022-8269 A, slow freezing, and vitrification. The above-mentioned freezing period and freezing method can also be applied to the step (d) of cryopreserving the cultured cells after step (b) in the method for producing the cell population.

[0070] The cryopreserved cell population of the present invention can also be banked in a cell bank. Taking the example of collecting mesenchymal cells from gingival tissue, specifically, when a patient's wisdom teeth are extracted and subsequently sutured, mesenchymal cells are collected and cultured (P0). The collected mesenchymal cells (P0) are then seeded, and the medium is changed one hour later, and the adhered cells are cultured. The cultured cells are detached from the medium, and the resulting cells (P1) can be cryopreserved and banked.

[0071] When utilized in regenerative medicine, the above-mentioned banked cryopreserved cells (P1) are seeded onto plates, cultured up to P4, and cryopreserved. When in use, they can be thawed and used for transplantation. The numbers in P0, P1, and P4 indicate the passage number.

[0072] The present disclosure will be explained in more detail below using examples, but the technical scope of the present disclosure is not limited to these examples.

[0073] Example 1 Preparation of Mesenchymal Cells Collected from Gingival Tissue First, gingival-derived cells were cultured by the following method to produce P1 cells, P2 cells, or P3 cells, which were used in the following examples. The numbers in P1, P2, and P3 indicate the passage number.

[0074] <Preparation of P1 cells> Gingival tissue from five donors (No. 1-5) was minced using tweezers and a scalpel, and the minced gingival tissue was attached to a 6-well plate (#MS-80060S: Sumitomo Bakelite Co., Ltd.). 1.5 mL of bovine-derived cell culture serum NeoSERA® (Japan Biomedical Co., Ltd.), 15 mL of AIM-V Medium (Thermo Fisher Scientific), and 15 mL of HFDM-1(+) (Cell Science Institute Co., Ltd.) were placed in one 50 mL tube, and the culture medium was prepared by pipetting. 5 mL of the culture medium was placed in each well of the 6-well plate, and the above-mentioned minced gingival tissue was cultured. P0 cells growing from the minced gingival tissue were collected and seeded in a 90 mm Petri dish (#MS-13900S: Sumitomo Bakelite Co., Ltd.). After 1 hour, the medium was changed and the adhered cells were cultured. The medium for one 90 mm Petri dish consisted of 4.5 mL of AIM-V Medium, 4.5 mL of HFDM-1(+), and 1 mL of NeoSERA. When the density of the cells cultured in the 90 mm Petri dish reached 70-95%, P1 cells were collected and cultured at a concentration of 1 x 10 in STEM-CELLBANKER® GMP grade (product code #CB045: Zenoac Resources Co., Ltd.). ６ The solution was adjusted to 100 cells / mL, dispensed in 1 mL aliquots into cryotubes to prepare cell preservation solutions containing P1 cells, and stored frozen at −80° C. until use.

[0075] <CD90 Expression in P1 Cells> The expression of CD90 in P1 cells was confirmed by the following method. P1 cells were diluted with PBS to 1 × 10 ６ The concentration was adjusted to 15 mL of cells / mL, and the 15 mL tube was placed on ice. 20 μL of each of the anti-CD90 antibodies or isotype control antibodies listed in Table 1 was placed in a 1.5 mL tube, and then the P1 cells (1 × 10 ６100 μL of PBS (100 μL / mL) was added and pipetted, and the tube was shielded from light with ice and allowed to stand for 30 minutes. In Table 1, ISO refers to isotype control. 400 μL of PBS was added to a 1.5 mL tube, pipetted, and passed through a 5 mL polystyrene round tube with a cell strainer and cap (#REF352235, Becton Dickinson). The tube was transferred to a PP standard test tube (round bottom) (#A26428, Beckman Coulter) and placed on ice until sample measurement began. The fluorescence wavelength of No. 1 was measured using a Novocyte (ACEA Biosciences), and the fluorescence wavelength of Nos. 2 to 5 was measured using a Cytomics FC 500 (Beckman Coulter).

[0076]

[0077] The results are shown in Table 2. From the results in Table 2, the obtained cell population contained 95% or more of CD90, a marker of mesenchymal cells, and it was determined that most of the obtained P1 cell population was mesenchymal cells. If the start of culturing the minced gingival tissue was considered to be day 0, the viable cell count of P1 cells on day 13 of culturing was 13.8 x 10 for each of Nos. 1 to 5. 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.

[0078]

[0079] <Preparation of P2 Cells> P2 cells were prepared in the following steps. The cell preservation solution containing the P1 cells dispensed into the cryotube was thawed. A 15 mL tube was charged with 3.5 mL of AIM-V Medium (Thermo Fisher Scientific), 0.5 mL of bovine cell culture serum NeoSERA (registered trademark) (Japan Biomedical Co., Ltd.), and 1 mL of the cell preservation solution containing the thawed P1 cells, and the tube was centrifuged at 1200 rpm for 1 minute at room temperature. The supernatant was removed by suction, and the P1 cells were seeded onto two 10 cm dishes (#93100: BM Instruments Co., Ltd.) and incubated in an incubator (37°C, 5% CO ２ ) and cultured for 4 days. The medium used for the 10 cm dish was 5 mL of AIM-V Medium + HFDM-1(+) (Cell Science Institute) + 1 mL of NeoSERA. The culture medium was aspirated from the 10 cm dish, and 5 mL of PBS (Cell Science Institute) was added to one 10 cm dish to wash the inside of the dish, after which the PBS was aspirated and removed. 2 mL of r-TE (Cell Science Institute), a recombinant trypsin / EDTA solution, was added to one 10 cm dish, and the 10 cm dish was placed in an incubator (37°C, 5% CO ２ ) and allowed to stand for 3 minutes. A 40 μm cell strainer (#352340: Corning) was attached to a 50 mL tube. Cells were detached from the 10 cm dish by pipetting and transferred to the 50 mL tube. 2 mL of s-TI (Cell Science Institute), a synthetic trypsin neutralizer solution, was added to one 10 cm dish to wash the inside of the 10 cm dish, and the washings were transferred to the 50 mL tube. The 50 mL tube was centrifuged at 1200 rpm for 1 minute at room temperature. After centrifugation, the supernatant was aspirated and the cells were suspended in 2 mL of AIM-V Medium to obtain a suspension containing P2 cells. 10 μL of the 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. For the doubling experiment described below, three cell culture flasks (#MS-23800 225 cm ２ : Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P2 cells ５The remaining cells were cultured in STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: Zenoac Resources) at a density of 1 x 10 P2 cells. ６ The solution was adjusted to 100 cells / mL, dispensed in 1 mL portions into cryotubes to prepare cell preservation solutions containing P2 cells, and stored frozen at −80° C. until use.

[0080] <Preparation of P3 cells> - For No. 1, P2 cells were seeded in a cell culture flask and passaged three days later, and for Nos. 2 to 5, P2 cells were seeded in a cell culture flask and passaged five days later. - For Nos. 2 to 5, half of the medium was replaced three days after cell seeding. - The culture medium was aspirated from one cell culture flask, and 25 mL of PBS was added to one cell culture flask to wash the inside of the cell culture flask, after which the PBS was aspirated. - 5 mL of r-TE (Cell Science Institute), a recombinant trypsin / EDTA solution, was added to one cell culture flask, and the cell culture flask was placed in an incubator (37°C, 5% CO ２ ) and allowed to stand for 3 minutes. A 40 μm cell strainer (#352340: Corning) was attached to a 50 mL tube. Cells were detached from the cell culture flask by pipetting and transferred to a 50 mL tube. 5 mL of s-TI (Cell Science Institute), a synthetic trypsin neutralizer solution, was added to one cell culture flask to wash the inside of the cell culture flask, and the washings were transferred to the 50 mL tube. The 50 mL tube was centrifuged at 1200 rpm for 2 minutes at room temperature. After centrifugation, the supernatant was aspirated and the cells were suspended in 20 mL of AIM-V Medium to obtain a suspension containing P3 cells. 10 μL of the cell suspension was mixed with 10 μL of trypan blue, and 10 μL of the mixture was applied to a hemocytometer to count the cell number. For the doubling experiment, three cell culture flasks (#MS-23800 225 cm ２ : Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P3 cells were added to each ５P3 cells were seeded at 1 x 10 in STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: Zenoac Resources). ６ The solution was adjusted to 100 cells / mL, dispensed in 1 mL aliquots into cryotubes to prepare cell preservation solutions containing P3 cells, and stored frozen at -80°C until use.

[0081] Example 2 Expression of Cell Surface Antigen Molecules in Mesenchymal Cells Collected from Gingival Tissue Among the numerous cell surface antigen molecules, the present inventors, based on their previous knowledge and know-how, focused on CD13, CD29, CD59, CD49e, CD151, CD280, CD304, CD274, CD63, CD106, as well as HLA class I and HLA class II, and used antibodies to examine the expression of these cell surface antigen molecules, along with cell surface molecules known as markers of mesenchymal stem cells.

[0082] One cryotube (cell preservation solution containing P3 cells) prepared in Example 1 was removed from -80°C and thawed. 3.5 mL of AIM-V Medium (Thermo Fisher Scientific), 0.5 mL of bovine-derived cell culture serum NeoSERA (registered trademark) (Japan Biomedical Co., Ltd.), and 1 mL of cell preservation solution containing thawed P3 cells were placed in a 15 mL tube, and the tube was centrifuged at 1200 rpm for 1 minute at room temperature. The supernatant was aspirated, and P3 cells were seeded onto two 10 cm dishes. The medium used for one 10 cm dish was 5 mL of AIM-V Medium + HFDM-1(+) (Cell Science Institute Co., Ltd.) + 1 mL of NeoSERA. The 10 cm dishes were then placed in an incubator (37°C, 5% CO ２ ) and cultured for 4 days. The culture medium was removed by suction from the 10 cm dish, and 5 mL of PBS (Cell Science Institute) was added to one 10 cm dish to wash the inside of the dish, after which the PBS was removed by suction. 2 mL of r-TE (Cell Science Institute), a recombinant trypsin / EDTA solution, was added to one 10 cm dish, and the 10 cm dish was placed in an incubator (37°C, 5% CO ２) and allowed to stand for 3 minutes. A 40 μm cell strainer (#352340: Corning) was attached to a 50 mL tube. The cells were detached from the 10 cm dish by pipetting and transferred to a 50 mL tube. 2 mL of s-TI (Cell Science Institute), a synthetic trypsin neutralizer solution, was added to one 10 cm dish to wash the inside of the 10 cm dish, and the washings were transferred to a 50 mL tube. The 50 mL tube was centrifuged at 1200 rpm for 1 minute at room temperature. After centrifugation, the supernatant was aspirated 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 and the cell number was counted. The cell concentration was 1 x 10 ６ After adjusting the concentration with PBS to 100 cells / mL, the 50 mL tube was placed on ice. 20 μL of each of the antibodies listed in Table 3 was placed in a 1.5 mL tube, and then the cell suspension (1 × 10 ６ 100 μL of PBS (100 μL / mL) was added and pipetted, and the tube was shielded from light with ice and allowed to stand for 30 minutes. 400 μL of PBS was added to a 1.5 mL tube and pipetted, then passed through a 5 mL polystyrene round tube with a cell strainer and cap (#REF352235: Becton Dickinson). The sample was transferred to a PP standard test tube (round bottom) (#A26428: Beckman Coulter) and placed on ice until sample measurement began. Fluorescence wavelength was measured using a Cytomics FC 500 (Beckman Coulter).

[0083]

[0084] The results are shown in Table 4. As is clear from Table 4, it was confirmed that in all cell populations obtained by culturing gingival tissue-derived mesenchymal cells Nos. 1 to 5, 95% or more of cells expressed CD13, CD29, CD59, CD49e, CD151, or CD280. Furthermore, in all cell populations obtained by culturing gingival tissue-derived mesenchymal cells Nos. 1 to 5, 97% or more of cells expressed CD90 or CD73, which are mesenchymal stem cell markers. Furthermore, the above cell populations contained 60% or more cells expressing CD274 and 16 to 66% cells expressing CD105. On the other hand, expression of CD34, CD45, CD56, CD304, or CD106 was not confirmed in any of the above cell populations, and only 0 to 0.4% of cells expressed HLA class II. Furthermore, expression of CD13, CD59, CD49e, CD151, and CD280 has not been confirmed in gingival-derived cell populations until now, and we have been able to identify new cell surface molecules in gingival-derived mesenchymal cells.

[0085]

[0086] [Example 3] Cell proliferation ability To evaluate the proliferation ability of cells, an experiment was conducted to measure the doubling time, which is the time it takes for a cell to double in size, i.e., the time it takes for one cell to divide into two.

[0087] The cell doubling time was measured by counting the number of cells per passage as follows: As described in Example 1 above, three cell culture flasks (#MS-23800 225 cm ２ (Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P2 cells prepared in Example 1 were added to each well. ５ After 5 days (2 days after medium change), the cells were collected and counted (P3 doubling experiment). Similarly, as described in Example 1 above, three cell culture flasks (#MS-23800 225 cm ２ (Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P3 cells prepared in Example 1 were added to each well. ５The cells were seeded, and 5 days later (2 days after medium change), the cells were collected and counted (P4 doubling experiment). Subsequently, the cells were subcultured and counted in the same manner as in Example 1 and described above to obtain passaged cells (P5 cells to P10 cells), confirm the cell count, and measure the doubling time.

[0088] The results are shown in Table 5. The numbers in Table 5 are doubling times (hr), with "P3" in the upper row indicating the doubling time from P2 to P3 ("P4" indicates the doubling time from P3 to P4; the same applies to P5 to P10). The doubling times for Nos. 2 to 5 were all within 35 hours, with P3, P4, and P5 in particular being within 26 hours. According to Table 1 of Non-Patent Document 6, the doubling time of cells is usually 25 to 53 hours. Therefore, it was confirmed that the cell populations of Nos. 2 to 5 proliferated in a very short period of time, with doubling times of 19 to 35 hours.

[0089]

[0090] Example 4 Cell Viability In cell transplantation therapy, isolated cells are expected to be cryopreserved and then thawed for use. It is necessary to maintain a high viability of the cryopreserved cells. Therefore, we investigated cell viability using cell populations obtained by culturing mesenchymal cells (four samples) collected from oral tissues.

[0091] Approximately 200 mL of blood was collected from patients (aged 40-80) with intractable skin ulcers caused by venous stasis, and autologous serum was prepared using CellAid (for serum collection, #JB-SB200CSA: JMS Co., Ltd.). Cells were collected from the tissue of the lower salivary gland (inside the cheek) of patients with intractable skin ulcers caused by venous stasis, and the tissue was shredded using tweezers and a scalpel. The shredded gingival tissue was then attached to a 6-well plate (#MS-80060S: Sumitomo Bakelite Co., Ltd.). 1.3 mL of autologous serum, 13 mL of AIM-V Medium (Thermo Fisher Scientific), 13 mL of HFDM-1(+) (Cell Science Institute), 130 μL of Meiji Collagenase G (#COLGS: Meiji Seika Pharma), and 52 μL of Meiji Collagenase H (#COLHS: Meiji Seika Pharma) were placed in one 50 mL tube and the culture medium was prepared by pipetting. 2 mL of the culture medium was placed in each well of a 6-well plate and cultured overnight. 1.5 mL of autologous serum, 15 mL of AIM-V Medium (Thermo Fisher Scientific), and 15 mL of HFDM-1(+) (Cell Science Institute) were placed in one 50 mL tube and pipetted to prepare the culture medium. The culture medium was removed from the wells of a 6-well plate, and 2 mL of the culture medium was placed in each well of the 6-well plate, and the above-mentioned shredded lower salivary gland (inside the cheek) tissue was cultured. Cells P0 emerging from the shredded tissue were collected and seeded onto a 90 mm Petri dish (#MS-13900S: Sumitomo Bakelite Co., Ltd.). After 1 hour, the medium was replaced and the adhered cells were cultured. The medium for one 90 mm dish consisted of 4.45 mL of AIM-V Medium, 4.45 mL of HFDM-1(+), and 0.5 mL of NeoSERA. When the density of the cells cultured in the 90 mm dish reached 70-95%, P1 cells were collected and transferred to a cell culture flask (#MS-23800S 225 cm ２ : Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P1 cells were added to each ５The cells were seeded and cultured. The culture medium for one cell culture flask was 25 mL of AIM-V Medium + 25 mL of HFDM-1(+) + 1 mL of autologous serum. The culture medium was aspirated from the cell culture flask, and 25 mL of PBS (Cell Science Institute) was added to one cell culture flask to wash the inside of the cell culture flask, after which the PBS was aspirated and removed. 7 mL of r-TE (Cell Science Institute), a recombinant trypsin / EDTA solution, was added to one cell culture flask, and the cell culture flask was placed in an incubator (37°C, 5% CO ２ ) and allowed to stand for 3 minutes. A 40 μm cell strainer (#352340: Corning) was attached to a 50 mL tube. Cells were detached from the cell culture flask by pipetting and transferred to the 50 mL tube. Seven mL of synthetic trypsin neutralizer solution, s-TI (Cell Science Institute), was added to one cell culture flask to wash the inside of a 10 cm dish, and the washings were transferred to the 50 mL tube. The 50 mL tube was centrifuged at 1200 rpm for 2 minutes at 20°C. After centrifugation, the supernatant was aspirated and the cells were suspended in 10 mL of HFDM-1(+) (Cell Science Institute) to obtain a suspension containing P2 cells. 10 μL of the 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. A portion of the P2 cells was cultured at 1 x 10 using STEM-CELLBANKER (registered trademark) GMP grade (product code #CB045: Zenoac Resources). ６ The solution was adjusted to 100 cells / mL, dispensed in 1 mL portions into cryotubes to prepare cell preservation solutions containing P2 cells, and stored frozen at −80° C. until use.

[0092] One of the cryotubes (cell preservation solution containing P2 cells) that had been frozen and stored at -80°C for a predetermined time was thawed, and the cell viability was measured using the following method. A 15 mL tube was charged with 8.5 mL of AIM-V Medium, 0.5 mL of NeoSERA, and the preservation solution containing the thawed P2 cells, and the tube was centrifuged (1200 rpm, 2 min, 20°C). The supernatant was removed by suction, and the cells were suspended in 2 mL of AIM-V Medium. The number of cells was counted in 10 μL of the tube using the same method as described in Example 1, and the P2 cell viability was measured. Cells were seeded onto two 10 cm dishes. 10 cm dish: AIM-V Medium 5 mL + HFDM-1(+) (Cell Science Institute) + NeoSERA 1 mL, cell suspension 1 mL. After 4 to 10 days of culture, the cells were detached from the 10 cm dish using r-TE / s-TI and centrifuged. The cells were then suspended in 2 mL of AIM-V Medium and counted in 10 μL to determine cell viability.

[0093] The results are shown in Table 6. Table 6 shows that 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%.

[0094]

[0095] [Example 5] Preparation of cell sheets Cell sheets were prepared using mesenchymal cells (No. 1 to No. 5) by the following method. For the doubling experiment in Example 1, three cell culture flasks (#MS-23800 225 cm ２ : Sumitomo Bakelite Co., Ltd.) 2.5 × 10 P3 cells were added to each ５ Cells were seeded in the flasks, and No. 1 was passaged 3 days after seeding, and Nos. 2 to 5 were passaged 5 days after seeding. During this passage, the cells were detached from the cell culture flask and counted. Then, P4 cells were seeded in the cell culture flask for passage, and 5 x 10 mesenchymal cells were seeded in one well of a 24-well plate. ５ The cells were seeded and cultured for 72 hours, and the supernatant was stored for ELISA analysis. Cell sheets were prepared from P4 cells by the method described in Patent Document 3, and the cell sheets were then transferred to a CellShifter to prevent shrinkage. ＴＭAfter adhering to the tissue, the tissue was fixed in 10% neutral formalin and then sectioned.

[0096] Figure 1 shows a photograph (4x magnification) of a section of the prepared cell sheet stained with hematoxylin and eosin (HE) and MT staining using standard methods. The HE staining in Figure 1 confirmed that the cells were organized into a cell sheet structure. Furthermore, MT staining confirmed that the cell sheet contained abundant extracellular matrix. Cell sheets could also be prepared using P3 cells and P5 to P10 cells (not shown).

[0097] Example 6: Expression of cell surface antigen molecules in cells collected from oral tissues. While cells collected from the gingiva were used in Example 2, cell surface antigen molecule analysis was performed in the same manner as in Example 2 using P4 cells (four specimens) collected from tissue from the lower salivary gland (inner cheek). The cells and culture method used were the same as in Example 4, and the cell surface antigen molecule analysis method, antibodies, and reagents used were the same as in Example 2. The results are shown in Table 7. It was confirmed that the cell population obtained from mesenchymal cells collected from tissue from the lower salivary gland (inner cheek) also contained 99% of cells expressing CD90, CD73, CD44, CD13, CD29, or CD59, 27% to 35% of CD105, 62% or more of CD166, 56% or more of CD49e, 93% or more of CD151, 97% or more of CD280, and 63% or more of CD63. However, no cells expressing CD34 or CD45 were detected.

[0098]

[0099] Example 7 Cell Surface Antigen Analysis of a Cell Population Cultured with IFN-γ-Treated Mesenchymal Cells Collected from Gingiva Cell surface antigen analysis (HLA class I, HLA class II, CD106, CD274) of a cell population cultured with IFN-γ-treated mesenchymal cells was performed according to the following procedure. One cryotube (cell preservation solution containing P3) prepared in Example 1 was removed from -80°C and thawed. A 15 mL tube was charged with 3.5 mL of AIM-V Medium, 0.5 mL of NeoSERA, and the thawed cell preservation solution, followed by centrifugation (1200 rpm, 1 min, 20°C). The supernatant was removed by suction, and the cells were seeded onto two 10 cm dishes and cultured for 3 days (37°C, 5% CO ２ ). The medium used for one 10 cm dish was AIM-V Medium 5 mL + HFDM-1(+) 5 mL, and NeoSERA 1 mL. After washing the cells with PBS, they were detached using r-TE / s-TI and centrifuged (1200 rpm, 1 min, 20°C), the supernatant was aspirated, and the cells were suspended in 2 mL of AIM-V Medium and counted using 10 μL. Each type of cell was seeded onto two 10 cm dishes and cultured overnight. 3 x 10 cells were placed on one 10 cm dish. ５ Cells were seeded. One 10 cm dish: 5.5 mL of AIM-V Medium + 4.5 mL of HFDM-1(+), 1 mL of NeoSERA. Ten 15 mL tubes were each filled with 4.5 mL of AIM-V Medium + 4.5 mL of HFDM-1(+), 1 mL of NeoSERA. Five of the above 15 mL tubes were each filled with 5 μL of 0.2 mg / mL R&D Recombinat Human IFN-r #285-IF / CF (dissolved in Otsuka distilled water (Otsuka Pharmaceutical Factory)) (final concentration: 100 ng / mL). Five other 15 mL tubes were each filled with 5 μL of Otsuka distilled water (Otsuka Pharmaceutical Factory). The culture medium was removed by aspiration from the 10 cm dish, and the above-mentioned adjusted medium was added and cultured for 48 hours, after which cell surface antigen analysis was performed.

[0100] Cell surface antigen analysis was performed in the same manner as in Example 2. The antibodies used were those listed in Table 3 above. The results are shown in Tables 8 and 9. Note that CD106 and C274 were experiments conducted independently, so they are shown in separate tables.

[0101] Table 8 confirms that IFN-γ treatment increases HLA class II expression. Furthermore, CD106 expression was not confirmed regardless of IFN-γ treatment. Furthermore, Table 9 confirms that IFN-γ treatment not only increases HLA class II expression, but also increases CD274 expression, resulting in a positive rate of 91 to 96%.

[0102]

[0103]

[0104] Although mesenchymal stem cells (MSCs) have low immunogenicity, it has been reported that they acquire immunogenicity in an inflammatory environment through the expression of HLA class II (MHC class II). Furthermore, cells expressing CD274 (PD-L1) have been reported to suppress the function of surrounding immune cells (Yu Yoshinaga et al., Stem Cell Reports. 2022;17:1714-1729). Therefore, in cell transplantation therapy using allogeneic cells, the expression of CD274 on transplanted allogeneic cells is thought to reduce the immunogenicity of the transplanted allogeneic cells by suppressing attack from the host's immune cells. While the percentage of cells expressing CD274 in the cell population established according to the present disclosure was 60% or more, culturing the cells in a medium supplemented with IFN-γ increased the percentage of cells expressing CD274 in the cell population to 91% or more. These results suggest that when a cell population obtained by culturing mesenchymal cells established according to the present disclosure is transplanted as allogeneic cells, it is possible to reduce the strong immune rejection reaction by suppressing attacks from immune cells in the host.

[0105] Example 8 VEGF Secretion VEGF secretion is important for use as a cell sheet. Therefore, VEGF secretion from cell sheets was investigated. The supernatant of the cell sheet prepared from mesenchymal cells collected from gingival tissue was the same supernatant stored for ELISA analysis in the preparation of the cell sheet in Example 5. The supernatant of the cell sheet prepared from mesenchymal cells collected from tissue of the lower salivary gland (inside the cheek) was prepared by the following method.

[0106] The P2 cell preservation solution prepared in Example 4 from mesenchymal cells collected from the tissue of the lower salivary gland (inside the cheek) was removed from −80°C and thawed. P3 cells were prepared from the P2 cells in the same manner as in Example 1 and placed in a 24-well plate (1.8 cm ２ / well, MS-80240: Sumitomo Bakelite Co., Ltd.) ５ pieces / cm ２ (The number of seeds in one well was 5.0 × 10 ５ pcs), incubator (37°C, 5% CO ２ After culturing for 72 hours, the culture medium was transferred to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes at 4°C, and the supernatant was stored at -80°C for ELISA analysis.

[0107] For ELISA analysis, the 1.5 mL tubes containing the supernatants for ELISA analysis were thawed on ice on the day of measurement, and the VEGF concentration in the supernatants was measured using the Human VEGF Quantikine ELISA Kit (R&D Systems, #DVE00). Absorbance measurements and concentration calculations were performed using an iMark Microplate Reader (BIO-RAD) and MPM6.exe (BIO-RAD).

[0108] (Results) The VEGF concentration in the medium when cell sheets were produced by culturing mesenchymal cells collected from the lower salivary gland tissue (inside the cheek) and mesenchymal cells collected from gingival tissue is shown in Figure 2. As shown in Figure 2, sufficient VEGF was secreted for both cell sheets, but it was confirmed that the cell sheet produced from mesenchymal cells collected from gingival tissue secreted at least twice as much VEGF as the cell sheet produced from mesenchymal cells collected from the lower salivary gland tissue (inside the cheek).

[0109] [Example 9] Presence or absence of SSEA-3 expression SSEA-3 is a cell surface antigen molecule known as a representative marker of pluripotent stem cells. We investigated whether gingival-derived mesenchymal cells express SSEA-3.

[0110] Using one cryotube (cell preservation solution containing P3 cells) prepared from gingival-derived mesenchymal cells from five donors Nos. 1 to 5 by the method described in Example 1, SSEA-3 expression was examined in the same manner as in Example 2. The antibodies used were an 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.

[0111] 3, there was no shift to the right of the peak, and the positive rate did not change, confirming that gingival-derived mesenchymal cells did not express SSEA-3.

Claims

1. A cell population isolated from a living body, comprising 80% or more mesenchymal cells that express 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 the mesenchymal cells are cells collected from oral tissue or a culture thereof.

3. The cell population according to claim 1 or 2, characterized in that 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.

4. The cell population according to claim 1 or 2, comprising 60% or more mesenchymal cells expressing CD274.

5. The cell population according to claim 1 or 2, comprising 5% or less mesenchymal cells expressing SSEA-3.

6. A three-dimensional cell culture comprising the cell population of claim 1 or 2.

7. A pharmaceutical composition comprising the cell population of claim 1 or 2.

8. The pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable additive.

9. The pharmaceutical composition of claim 7 in the form of a three-dimensional cell culture.

10. The pharmaceutical composition according to claim 7 , which is in the form of a cell sheet.

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

12. (a) collecting mesenchymal cells from biological tissue; (b) culturing the collected mesenchymal cells; 3. A method for producing the cell population described in claim 1 or 2, comprising the steps of:

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

14. The method according to claim 12, wherein in step (b), the cells are cultured in a medium containing an inflammatory cytokine.

15. Further, after step (b), (d) cryopreserving the cultured cells; 13. The method of claim 12, comprising:

Images