Articular cartilage treatment agent

Abstract

To provide a highly safe articular cartilage treatment agent.SOLUTION: An articular cartilage treatment agent contains scaffold-free artificial tissue where synovium-derived mesenchymal stem cells cultured serum-free form a three-dimensional structure.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for treating articular cartilage. [Background technology]

[0002] Articular cartilage plays a role in smooth joint movement and absorbing shock when loaded, and damage to it can cause movement disorders and pain, which tend to have a serious impact on daily life. In recent years, regenerative medicine using cell transplantation has been applied to treat such damage to articular cartilage.

[0003] As cells applicable to regenerative medicine, for example, Patent Document 1 proposes a scaffold-free self-organizing three-dimensional synthetic tissue that has tissue strength suitable for clinical application. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2005 / 012512 Brochure Summary of the Invention [Problem to be solved by the invention]

[0005] A high level of safety and quality is required for cells used in regenerative medicine, and there is room for improvement in the artificial tissue described in Patent Document 1. An object of one aspect of the present invention is to provide a highly safe therapeutic agent for articular cartilage. [Means for solving the problem]

[0006] In order to solve the above problems, an articular cartilage treatment agent according to one embodiment of the present invention contains a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in a serum-free environment form a three-dimensional structure. Effect of the Invention

[0007] Advantageous Effects of Invention One aspect of the present invention can provide a highly safe agent for treating articular cartilage. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the evaluation criteria for the cartilage regeneration function of an articular cartilage therapeutic agent used in the Examples. [Diagram 2] FIG. 1 shows the results of evaluation of the cartilage regeneration function of an articular cartilage treatment agent in an Example. [Diagram 3] FIG. 1 shows the results of histopathological examination in an example. [Figure 4] FIG. 1 shows the results of measuring cartilage-related gene expression in an example. [Diagram 5] FIG. 2 shows the results of measuring the expression levels of cartilage regeneration-related factors in culture supernatants in an example. [Figure 6] FIG. 2 shows the results of measuring the expression levels of cartilage regeneration-related factors in a gMSC (registered trademark)1 extract in an Example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The following describes an embodiment of the present invention, but the present invention is not limited thereto. In this specification, unless otherwise specified, "A to B" indicating a numerical range means "A or more and B or less."

[0010] [Articular cartilage treatment agent] An articular cartilage treatment agent according to one embodiment of the present invention contains a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in a serum-free environment form a three-dimensional structure.

[0011] The present inventors have found that a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum form a three-dimensional structure is effective for treating articular cartilage. The present inventors have also found that a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum form a three-dimensional structure has good engraftment at the transplant site and is suitably differentiated into chondrocytes. As a result, an articular cartilage treatment agent containing a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum form a three-dimensional structure has excellent therapeutic effects.

[0012] The articular cartilage therapeutic agent can be used to treat a damaged or defective site of articular cartilage. The articular cartilage therapeutic agent treats damage or defects in articular cartilage by administering it to a site requiring treatment. By administering the articular cartilage therapeutic agent to a damaged or defective site of articular cartilage, regeneration of articular cartilage at the administration site is promoted, and damage or defects in articular cartilage can be treated. The articular cartilage therapeutic agent can be used to treat cartilage in any joint, such as a knee joint, elbow joint, or shoulder joint. The articular cartilage treated by the articular cartilage therapeutic agent may include meniscus tissue.

[0013] In articular cartilage treatment using an articular cartilage therapeutic agent, the method of administering the articular cartilage therapeutic agent is not particularly limited, and examples include a method of transplanting the articular cartilage therapeutic agent into the treatment site, injecting an injection solution containing the articular cartilage therapeutic agent into the treatment site, etc. The articular cartilage therapeutic agent may be administered to the treatment site alone or in combination with other therapeutic agents.

[0014] (Mesenchymal stem cells derived from synovium) In this specification, "mesenchymal stem cells" refers to somatic stem cells that differentiate into tissues belonging to the mesenchymal system. In addition, mesenchymal stem cells also include those isolated from mesenchymal stem cells with further specific properties, those given some kind of stimulation such as cytokine stimulation to mesenchymal stem cells, and those that have been genetically introduced into mesenchymal stem cells. For example, MUSE cells, MAPC cells, SP-1 cells, etc. are also included in mesenchymal stem cells. Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes, etc. Mesenchymal stem cells are known to be isolated not only from adult tissues such as bone marrow, fat, synovium, alveolar bone, and periodontal ligament, but also from placenta, umbilical cord, umbilical cord blood, various fetal tissues, etc.

[0015] The synovial-derived mesenchymal stem cells forming the artificial tissue contained in the articular cartilage therapeutic agent are stem cells contained in the synovial membrane, and are obtained from synovial tissue by a known method. The synovial-derived mesenchymal stem cells have the ability to differentiate into chondrocytes. The synovial-derived mesenchymal stem cells may be mesenchymal stem cells derived from the synovial membrane of non-human animals such as rats and mice, but are preferably mesenchymal stem cells derived from human synovial membrane. When the articular cartilage therapeutic agent is administered to a human treatment site, the human synovial-derived mesenchymal stem cells have better engraftment at the treatment site and have excellent therapeutic effects. In addition, the human synovial-derived mesenchymal stem cells can reduce the risk of biological contamination and the presence of immunogenic substances in the human to whom the articular cartilage therapeutic agent is administered.

[0016] The synovium-derived mesenchymal stem cells are cultured without serum. In serum-free culture, the culture components are known. In other words, since serum is derived from natural components, there are differences in the components from lot to lot, but such differences do not occur in serum-free media. Therefore, synovium-derived mesenchymal stem cells cultured without serum are excellent in both safety and quality. In addition, it is possible to minimize the risks of biological contamination and the presence of immunogenic substances, and to minimize substances that do not exist in the body. In addition, since the biological raw materials contained are also clear, quality control is easy. In addition, mesenchymal stem cells cultured without serum in some serum-free media such as STK (registered trademark) medium have excellent proliferation rates.

[0017] As used herein, the term "serum-free culture" refers to culture without using serum. For example, culture using a serum-free medium that does not contain serum is intended.

[0018] (serum-free culture) An example of a serum-free medium used for serum-free culture of synovial-derived mesenchymal stem cells forming artificial tissue contained in an articular cartilage treatment agent will be described. The basal medium for constituting the serum-free medium is not particularly limited as long as it is a medium for animal cells well known in the art, and preferred basal media include, for example, Ham's F12 medium, DMEM medium, RPMI-1640 medium, and MCDB medium. These basal media may be used alone or in combination. In one embodiment, the basal medium for constituting the serum-free medium is preferably a medium in which MCDB and DMEM are mixed in a ratio of 1:1.

[0019] In one embodiment, a serum-free medium obtained by adding FGF, PDGF, TGF-β, HGF, EGF, at least one phospholipid, and at least one fatty acid to the above-mentioned basal medium may be used for culturing mesenchymal stem cells derived from synovium. The content of FGF in the basal medium is preferably 0.1 to 100 ng / ml, more preferably 3 ng / ml, in terms of final concentration. The content of PDGF in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, in terms of final concentration. The content of TGF-β in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, in terms of final concentration.

[0020] The content of HGF in the basal medium is preferably 0.1 to 50 ng / ml, more preferably 5 ng / ml, in terms of final concentration. The content of EGF in the basal medium is preferably 0.5 to 200 ng / ml, more preferably 20 ng / ml, in terms of final concentration. The total content of phospholipids in the basal medium is preferably 0.1 to 30 μg / ml, more preferably 10 μg / ml, in terms of final concentration. The total content of fatty acids in the basal medium is preferably 1 / 1000 to 1 / 10 of the basal medium, more preferably 1 / 100.

[0021] By using such a serum-free medium, it is possible to obtain a proliferation-promoting effect equal to or greater than that of a serum-containing medium while preventing contamination with foreign proteins, thereby enabling synovial membrane-derived mesenchymal stem cells to proliferate as desired.

[0022] The serum-free medium may contain phospholipids. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol, and these phospholipids may be contained alone or in combination. In one embodiment, the serum-free medium may contain phosphatidic acid and phosphatidylcholine in combination, and these phospholipids may be derived from animals or plants.

[0023] The serum-free medium may contain a fatty acid. Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid, and the medium additive according to the present embodiment may contain these fatty acids alone or in combination. The serum-free medium according to the present embodiment may further contain cholesterol in addition to the above fatty acids.

[0024] As used herein, FGF refers to a growth factor selected from the fibroblast growth factor family, preferably FGF-2 (bFGF), but may be selected from other FGF families such as FGF-1. As used herein, PDGF refers to a growth factor selected from the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB. As used herein, TGF-β refers to a growth factor selected from the transforming growth factor-β family, preferably TGF-β1, but may be selected from other TGF-β families.

[0025] As used herein, by HGF is intended a growth factor selected from the hepatocyte growth factor family, and by EGF is intended a growth factor selected from the epidermal growth factor family.

[0026] In one embodiment, the serum-free medium may further contain at least two factors selected from the group consisting of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and an ascorbic acid compound.

[0027] As used herein, an ascorbic acid compound is intended to mean ascorbic acid (vitamin C) or ascorbic acid 2-phosphate, or compounds similar thereto.

[0028] The above-mentioned growth factors contained in the serum-free medium may be natural growth factors or those produced by genetic recombination.

[0029] In one aspect, the serum-free medium preferably contains a lipid oxidation inhibitor. In one embodiment, the lipid oxidation inhibitor contained in the serum-free medium may be DL-α-tocopherol acetate (vitamin E). The serum-free medium may further contain a surfactant. In one embodiment, the surfactant contained in the serum-free medium may be Pluronic F-68 or Tween 80.

[0030] The serum-free medium may further contain insulin, transferrin and selenate. As used herein, insulin may be an insulin-like growth factor, and may be derived from natural cells or produced by genetic recombination. The medium additive of the present invention may further contain dexamethasone or other glucocorticoids.

[0031] When culturing synovial-derived mesenchymal stem cells in a serum-free medium, mesenchymal stem cells isolated from the synovial tissue of animals such as humans by a conventional method are seeded in the above-mentioned serum-free medium and cultured until they proliferate to the desired number. 4 It is preferable to seed individual mesenchymal stem cells at a culture temperature of 37°C ± 1°C for a culture time of 48 to 96 hours under 5% CO. By culturing in this manner, it is possible to efficiently obtain large amounts of mesenchymal stem cells that maintain or improve their immunosuppressive ability.

[0032] The culture vessel used for the culture is not particularly limited as long as it is capable of growing mesenchymal stem cells. For example, a 75 cm 2 Flask, Sumitomo Bakelite 75cm 2 Flasks and the like can be suitably used. However, depending on the cells, the proliferation of the cells may be affected by the type of culture vessel used. Therefore, in order to proliferate synovial membrane-derived mesenchymal stem cells more efficiently, it is preferable to culture each mesenchymal stem cell to be proliferated (hereinafter also referred to as "proliferation target cells") in a culture vessel suitable for proliferation.

[0033] As a method for selecting a culture vessel suitable for the proliferation of the proliferation target cells, for example, a method of having the proliferation target cells select the optimal culture vessel can be mentioned. Specifically, a plurality of types of culture vessels are prepared, the proliferation target cells are proliferated under the same culture conditions except for the types of culture vessels, and the number of cells two weeks after the start of the culture is measured by a known method, and the culture vessels with the largest number of cells can be judged as being suitable for the proliferation of the proliferation target cells. In addition, if the proliferation rate of the proliferation target cells is high, the culture vessels with the shortest period of time to reach 80 to 90% of the cell number in the confluent state can be judged as being suitable for the proliferation of the proliferation target cells, even before two weeks have passed since the start of the culture.

[0034] In addition, since the proliferation of mesenchymal stem cells requires that the cells adhere to the culture vessel, if the adhesion of the cells to be proliferated to the culture vessel is weak, it is preferable to further include a cell adhesion molecule in the serum-free medium when carrying out serum-free culture. Examples of the cell adhesion molecule include fibronectin, collagen, gelatin, etc. These cell adhesion molecules may be used alone or in combination.

[0035] The content of the cell adhesion molecule in the serum-free medium is preferably 1 to 50 μg / ml, more preferably 5 μg / ml, in terms of final concentration. In one embodiment, when fibronectin is used as the cell adhesion molecule, the adhesion efficiency of the target cells to be grown on the culture vessel can be improved by adding fibronectin to the serum-free medium so that the final concentration of the fibronectin becomes 5 μg / ml.

[0036] In addition, in serum-free culture, synovial-derived mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells grow in an anchorage-dependent manner, in cases where mesenchymal stem cells grow unevenly in a localized area, the culture conditions can be improved by passage of synovial-derived mesenchymal stem cells during the growth process.

[0037] The method of passage of synovial-derived mesenchymal stem cells is not particularly limited, and they can be passaged using a conventionally known method of passage of mesenchymal stem cells. Since the condition of synovial-derived mesenchymal stem cells after passage is good, it is preferable to use a cell detachment agent that does not contain mammalian and microbial components to detach the mesenchymal stem cells when passage is performed. Examples of the above-mentioned "cell detachment agent that does not contain mammalian and microbial components" include TrypLE Select CTS (Thermo Fisher Scientific Inc.) and ACCUTASE (Innovative Cell Technologies, Inc.).

[0038] (artificial tissue) The artificial tissue contained in the articular cartilage therapeutic agent according to one embodiment of the present invention is a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured without serum form a three-dimensional structure. The artificial tissue may be a tissue engineered construct (TEC). When cells are transplanted by administering a cell suspension to an affected area, it has been reported that the administered cells tend to detach from the transplant site and do not remain at the transplant site. However, since the articular cartilage therapeutic agent according to one embodiment of the present invention contains a scaffold-free artificial tissue with a three-dimensional structure, the cells tend to take root at the transplant site.

[0039] In addition, a scaffold-free artificial tissue having a three-dimensional structure may be prepared by processing a cell mass of mesenchymal stem cells into a three-dimensional structure by a conventionally known method. In this specification, the term "three-dimensional structure" refers to an object that extends in a three-dimensional direction, in which the matrix is ​​three-dimensionally oriented, the cells are three-dimensionally arranged, and the cells maintain their intercellular bonds and orientation.

[0040] The area, thickness, and strength of the artificial tissue may be appropriately set to be suitable for the treatment of articular cartilage, and those skilled in the art can appropriately set the size. The size can be set according to the environment in which it is to be transplanted. An artificial tissue with a small size has the advantage that it can be injected into a body cavity with a syringe needle. In addition, an artificial tissue with a large size has the advantage that it is easy to handle, for example, to be easily grasped with pins during surgery, and therefore a sufficient number of cells can be easily administered.

[0041] When the artificial tissue is to be implanted, it is preferable that the artificial tissue has at least a certain size. Such a size is, for example, 1 cm 2 for the area of ​​the artificial tissue that has formed a three-dimensional structure. 2 More than 2 cm, preferably 2 More preferably, 3 cm 2 More preferably, it is 4 cm. 2 More than 5cm 2 More than 6cm 2 More than 7cm 2 More than 8cm 2 More than 9cm 2 More than 10cm 2 More than 15cm 2 or more, or 20cm 2 or more, for example, 40 cm 2 Below, 30cm 2 Below, 20cm 2 The area may be, but is not limited to, 1 cm or less depending on the application. 2 Less than or equal to 40cm 2 It could be more than that.

[0042] When expressed in terms of the volume of the artificial tissue, the above size is preferably 2 mm 3 More preferably, it is 40 mm or more. 3 or more, for example, 40 cm 3 Less than or equal to 20cm 3 It may be, but is not limited to, 2 mm 3 It could also be the following.

[0043] The sufficient thickness of the implantable artificial tissue varies depending on the part to be implanted, but those skilled in the art can set the thickness appropriately. This thickness can be set according to the environment to be implanted. It is intended that the thickness of the artificial tissue is 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. When the artificial tissue is applied to cartilage, it may be, for example, 1 mm or more, preferably 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. In either case, it may be 1 mm or less, 10 mm or less, or 5 mm or less.

[0044] The number of cells constituting the artificial tissue may be appropriately selected, and may be, for example, a mass of 50 to 200 cells, or a mass of 1 million to 100 million cells. Furthermore, the mass may be small or large.

[0045] (Scaffold-free) As used herein, "scaffold-free" refers to substantially no materials (base materials = scaffolds) that are conventionally used in producing artificial tissues. Examples of such scaffold materials include, but are not limited to, chemical polymer compounds, ceramics, and biological preparations such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold refers to a material that is substantially solid and has the strength to support cells or tissues.

[0046] Conventionally, the mainstream cell preparations are "scaffold-type" cell preparations, which are processed into three-dimensional structures by artificially adding a material that serves as a scaffold for cells and tissues to attach or hold cells and enable their growth. However, recently, due to concerns about the risk of artificially adding materials, development of "scaffold-free" cell preparations has been promoted, which are produced without artificially adding a scaffold, for example, by stimulating the cells themselves to generate an environment that serves as their own scaffold.

[0047] Since the artificial tissue is a scaffold-free three-dimensional structure, the amount of materials other than mesenchymal stem cells contained in the articular cartilage treatment agent can be reduced. Furthermore, since the artificial tissue is a scaffold-free three-dimensional structure, the components are known, the risks of biological contamination and the presence of immunogenic substances can be minimized, and substances that do not exist in the body can be minimized. For example, natural products such as collagen are sometimes used as scaffolds. The components of such natural products vary from lot to lot. However, since the scaffold-free three-dimensional structure is known, the components are excellent in safety and quality stability. Furthermore, such natural products have the risk of biological contamination and the presence of immunogenic substances. Since the artificial tissue is a scaffold-free three-dimensional structure, such risks can be reduced.

[0048] As a method for obtaining a scaffold-free artificial tissue, which is a three-dimensional structure containing serum-free cultured synovium-derived mesenchymal stem cells contained in the articular cartilage treatment agent according to one embodiment of the present invention, for example, a method using a conventionally known low-adhesion plate, a micropattern surface plate, etc., and the hanging drop method can be adopted. In addition, an artificial tissue may be prepared using the method described in Japanese Patent No. 4522994. In addition, a commercially available product may be used, and for example, gMSC (registered trademark) 1 (manufactured by Two Cells Co., Ltd.) can be preferably used.

[0049] (Extracellular Matrix) The articular cartilage therapeutic agent according to one embodiment of the present invention may further contain an extracellular matrix derived from mesenchymal stem cells. As used herein, the term "extracellular matrix" refers to a substance present between somatic cells, whether epithelial cells or non-epithelial cells, also known as extracellular matrix.

[0050] Extracellular matrix is ​​one of the biological materials produced by cells, and is known to be involved not only in supporting tissues but also in forming the internal environment necessary for the survival of all somatic cells. Representative extracellular matrices include, but are not limited to, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), and various extracellular matrices that are responsible for cell adhesion can be used in the present invention.

[0051] The extracellular matrix preferably includes at least one selected from the group consisting of collagen, vitronectin, and fibronectin. In the articular cartilage therapeutic agent, the extracellular matrix may be integrated with the artificial tissue to form a three-dimensional structure, or may be present in the articular cartilage therapeutic agent independently of the artificial tissue. The articular cartilage therapeutic agent may further include a reagent, a buffer, etc. for stably maintaining the artificial tissue.

[0052] (Composition for cryopreservation) The articular cartilage therapeutic agent according to one embodiment of the present invention may further comprise a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters. In the present specification, the "cryopreservation composition" refers to a cryopreservation composition for cryopreserving a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in a serum-free environment form a three-dimensional structure. The articular cartilage therapeutic agent further comprises a cryopreservation composition, and can be cryopreserved, and is well frozen with a good recovery rate and survival rate, and the recovery rate and survival rate after cryopreservation and thawing are also good. In addition, the quality of the artificial tissue after thawing of the cryopreserved articular cartilage therapeutic agent is normal, and the properties of the artificial tissue do not change even after cryopreservation and thawing. Therefore, it can be cryopreserved until it is needed and used when needed.

[0053] Examples of fatty acids contained in the cryopreservation composition include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Among them, it is preferable that at least one of linoleic acid and linolenic acid is contained in the cryopreservation composition. By containing at least one of linoleic acid and linolenic acid in the cryopreservation composition, the cryopreservation composition can freeze mesenchymal stem cells with a higher survival rate. In addition, it may be a short-chain fatty acid, a medium-chain fatty acid, or a long-chain fatty acid. Furthermore, it may be a highly unsaturated fatty acid. These may be used alone or in a mixture of multiple types. Among them, a mixture of multiple fatty acids is preferable, and a mixture in which the type and amount of the fatty acid contained is specified is more preferable. For example, Chemically defined lipid concentrate (manufactured by Thermo Fisher Scientific Inc., product number 11905-031) is more preferable.

[0054] As described above, the fatty acids exemplified above may be used alone or in combination, but it is more preferable to use a mixture of multiple types of fatty acids. By including a mixture of more types of fatty acids in the cryopreservation composition, the artificial tissue can be frozen with a higher survival rate, and the properties of the artificial tissue do not change after thawing.

[0055] The content of fatty acid in the cryopreservation composition is not particularly limited, but for example, the final concentration is preferably 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreservation composition, and more preferably 1 / 1000 to 1 / 10 (v / v) relative to the total amount of the cryopreservation composition.

[0056] Examples of fatty acid esters contained in the cryopreservation composition include phospholipids and neutral fats. Among them, it is preferable that the cryopreservation composition contains phospholipids. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.

[0057] When a fatty acid ester such as a phospholipid is used, its content is preferably such that the final concentration in the cryopreservation composition at the time of use is 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreservation composition.

[0058] The cryopreservation composition may further contain a substance that aids in the dissolution (emulsification) of the fatty acid or fatty acid ester in water, such as a surfactant, which may be Pluronic F-68 or Tween 80.

[0059] The components of the cryopreservation composition may contain a cryoprotectant that inhibits the growth of ice crystals in cells during the freezing and thawing process. Examples of cryoprotectants include DMSO (dimethyl sulfoxide). When the cryopreservation composition contains a cryoprotectant, the amount of the cryoprotectant is more preferably 0.5% to 50% (v / v) of the total amount of the cryopreservation composition.

[0060] The cryopreservation composition may further contain other components, such as, for example, a basal medium, a viscosity increasing agent, a pH adjusting agent, and a cryoprotectant.

[0061] Moreover, the cryopreservation composition more preferably contains insulin, albumin, and transferrin. Insulin, albumin, and transferrin can enhance the action of fatty acids. When these are added, the concentration is preferably such that the final concentration in the cryopreservation composition at the time of use is 0.5 μg / ml to 500 μg / ml.

[0062] The articular cartilage treatment agent can be cryopreserved by a method including a step of immersing a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum form a three-dimensional structure in a solution of a cryopreservation composition, and a step of freezing the artificial tissue in the solution. The freezing temperature may be appropriately set to a temperature at which the artificial tissue to be frozen freezes, for example, -80°C or lower, or -196°C or lower. When freezing at -80°C or lower, for example, a conventionally known example may be used, and when freezing at -196°C or lower, liquid nitrogen may be used.

[0063] The cryopreserved articular cartilage therapeutic agent can be used by melting at a temperature that does not damage the scaffold-free artificial tissue in which synovial membrane-derived mesenchymal stem cells that have been cultured in a serum-free environment form a three-dimensional structure. Examples of the melting method include general methods such as a water bath melting method, a heat block method, and a room temperature melting method.

[0064] The melting temperature is preferably 10°C or higher and 45°C or lower, and more preferably 20°C or higher and 40°C or lower. For example, the mixture may be left to stand at room temperature (25°C), but it is more preferable to melt the mixture in a water bath at 35 to 38°C, as shown in the examples.

[0065] [Method of evaluating articular cartilage treatment agent] A method for evaluating an articular cartilage therapeutic agent according to one embodiment of the present invention comprises the step of detecting the presence of a mixture of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage therapeutic agent according to one embodiment of the present invention has been administered. According to the method for evaluating an articular cartilage therapeutic agent, the therapeutic effect of the articular cartilage therapeutic agent on articular cartilage is evaluated using as an indicator the differentiation of mesenchymal stem cells contained in the articular cartilage therapeutic agent into articular chondrocytes at the site where the articular cartilage therapeutic agent has been administered.

[0066] In the evaluation step, by detecting the presence of a mixture of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage therapeutic agent was administered, it is possible to detect that the administered mesenchymal stem cells have differentiated into articular chondrocytes. Furthermore, by detecting that the administered mesenchymal stem cells have differentiated into articular chondrocytes, it is possible to evaluate that the articular cartilage therapeutic agent functions in treating joints.

[0067] The coexistence of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage therapeutic agent is administered can be detected by double staining of mesenchymal stem cells and articular chondrocytes. Double staining of mesenchymal stem cells and articular chondrocytes can be performed, for example, by staining mesenchymal stem cells for human vimentin and staining articular chondrocytes for type II collagen. Staining of human vimentin and staining of type II collagen can be performed by conventionally known methods.

[0068] In the evaluation step, if the double staining results in detection of only stained human vimentin at the site where the articular cartilage therapeutic agent was administered, it can be determined that mesenchymal stem cells and articular chondrocytes are not mixed together, and as a result, it can be evaluated that the administered mesenchymal stem cells have not differentiated into articular chondrocytes and that the articular cartilage therapeutic agent has not functioned in treating the joint.

[0069] Furthermore, if the double staining results in the detection of both stained human vimentin and type II collagen at the site where the articular cartilage therapeutic agent was administered, it can be assessed that the administered mesenchymal stem cells have differentiated into articular chondrocytes and that the articular cartilage therapeutic agent is functioning in treating the joint.

[0070] In the evaluation step, it is preferable to detect the presence of stained type II collagen in the stained human vimentin region. In this way, by detecting the overlap between the human vimentin region and the type II collagen region, it is possible to more reliably evaluate that the administered mesenchymal stem cells have differentiated into articular chondrocytes. In addition, when type II collagen is stained in such an overlapping region with the same intensity as normal articular chondrocytes, it can also be evaluated that the joint treatment function of the articular cartilage treatment agent is superior.

[0071] According to the method for evaluating an articular cartilage therapeutic agent, it is possible to accurately evaluate the regeneration of articular cartilage by the administered articular cartilage therapeutic agent. According to the method for evaluating an articular cartilage therapeutic agent, it is possible to distinguish and evaluate natural healing at the damaged or defective site of articular cartilage from treatment by the articular cartilage therapeutic agent. As a result, in addition to evaluating the therapeutic effect of the articular cartilage therapeutic agent, it is possible to evaluate the regeneration mechanism of articular cartilage by the articular cartilage therapeutic agent.

[0072] The subject of evaluation by the evaluation method for an articular cartilage therapeutic agent may be an animal including a human, but may also be an animal other than a human. If the animal other than a human is, for example, a non-human mammal, it is possible to evaluate to some extent the therapeutic effect of the articular cartilage therapeutic agent on humans from the evaluation results of the animal.

[0073] [Method of Treating Articular Cartilage] The method for treating articular cartilage according to one aspect of the present invention comprises administering a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum form a three-dimensional structure. The mesenchymal stem cells forming the artificial tissue administered in the method for treating articular cartilage may be derived from human synovium. Furthermore, the artificial tissue administered in the method for treating articular cartilage may further contain an extracellular matrix derived from mesenchymal stem cells. That is, since one aspect of the artificial tissue in the method for treating articular cartilage is the artificial tissue contained in the articular cartilage therapeutic agent according to one aspect of the present invention, the explanation of the artificial tissue is as referred to in the explanation of the artificial tissue in the articular cartilage therapeutic agent.

[0074] In the administration step, the artificial tissue can be administered by transplanting the artificial tissue to the treatment site, injecting an injection solution containing the artificial tissue to the treatment site, etc. In the administration step, the artificial tissue may be administered alone or in combination with other therapeutic agents.

[0075] The dosage of the artificial tissue in the administration step can be appropriately determined by those skilled in the art, taking into consideration the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the form or type of tissue. The administration frequency of the artificial tissue in the administration step can also be appropriately determined by those skilled in the art, taking into consideration the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the course of treatment. Examples of the administration frequency include administration every day to once every few months (for example, once a week to once a month). The dosage and administration frequency of the artificial tissue in the administration step may be appropriately adjusted depending on the course of treatment.

[0076] The treatment site to which the artificial tissue is administered in the administration step is a damaged or defective site of articular cartilage. By administering the artificial tissue to a damaged or defective site of articular cartilage, regeneration of the articular cartilage is promoted, and the damage or defect of the articular cartilage can be treated.

[0077] Mesenchymal stem cells for use in the treatment of articular cartilage The mesenchymal stem cells according to one embodiment of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are derived from synovium and cultured in a serum-free manner, and form a three-dimensional scaffold-free artificial tissue. The mesenchymal stem cells for use in the treatment of articular cartilage may be derived from human synovium. Furthermore, the mesenchymal stem cells for use in the treatment of articular cartilage may further contain an extracellular matrix derived from mesenchymal stem cells. That is, since the mesenchymal stem cells for use in the treatment of articular cartilage are mesenchymal stem cells that form the artificial tissue contained in the articular cartilage treatment agent according to one embodiment of the present invention, the explanation of the mesenchymal stem cells is based on the explanation of the mesenchymal stem cells and the artificial tissue in the articular cartilage treatment agent.

[0078] Mesenchymal stem cells for use in the treatment of articular cartilage can be used to treat damaged or defective sites of articular cartilage. Mesenchymal stem cells for use in the treatment of articular cartilage are administered to a site in need of treatment to treat damaged or defective articular cartilage. By administering mesenchymal stem cells for use in the treatment of articular cartilage to a damaged or defective site of articular cartilage, regeneration of articular cartilage at the administration site is promoted, and damaged or defective articular cartilage can be treated.

[0079] [Use of mesenchymal stem cells for producing articular cartilage treatment agent] The use of mesenchymal stem cells for producing an articular cartilage therapeutic agent according to one embodiment of the present invention is a use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, in which the mesenchymal stem cells are derived from synovium and cultured in a serum-free environment to form a three-dimensional scaffold-free artificial tissue. In addition, in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, the mesenchymal stem cells may be derived from human synovium. Furthermore, in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, the artificial tissue may further contain an extracellular matrix derived from mesenchymal stem cells. That is, one embodiment of the artificial tissue in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent is the artificial tissue contained in the articular cartilage therapeutic agent according to one embodiment of the present invention, and therefore the description of the artificial tissue is the same as that of the artificial tissue in the articular cartilage therapeutic agent.

[0080] The articular cartilage therapeutic agent produced by using mesenchymal stem cells may further include a pharma- tically acceptable carrier and the like, together with a scaffold-free artificial tissue in which serum-free cultured synovium-derived mesenchymal stem cells form a three-dimensional structure. The pharma- tically acceptable carrier contained in the articular cartilage therapeutic agent may include any substance known in the art. The pharma- tically acceptable carrier may include, but is not limited to, antioxidants, preservatives, colorants, flavorings, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0081] An articular cartilage therapeutic agent produced by using mesenchymal stem cells can be used to treat damaged or defective sites of articular cartilage. The articular cartilage therapeutic agent treats damage or defects of articular cartilage by administering it to a site requiring treatment. By administering the articular cartilage therapeutic agent to a damaged or defective site of articular cartilage, regeneration of articular cartilage at the administration site is promoted, and damage or defects of articular cartilage can be treated.

[0082] 〔summary〕 The articular cartilage treatment agent according to aspect 1 of the present invention contains a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in a serum-free environment form a three-dimensional structure.

[0083] An articular cartilage therapeutic agent according to a second aspect of the present invention is in accordance with the first aspect, wherein the mesenchymal stem cells are derived from human synovium.

[0084] The articular cartilage treatment agent according to aspect 3 of the present invention, in accordance with aspect 1 or 2, may further contain an extracellular matrix derived from mesenchymal stem cells.

[0085] The articular cartilage treatment agent of aspect 4 of the present invention, in any one of aspects 1 to 3, may further comprise a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters, and may be cryopreserved.

[0086] A method for evaluating an articular cartilage therapeutic agent according to aspect 5 of the present invention comprises the step of detecting the presence of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage therapeutic agent of any of aspects 1 to 4 has been administered.

[0087] A method for treating articular cartilage according to a sixth aspect of the present invention comprises the step of administering a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells have formed a three-dimensional structure by serum-free culture.

[0088] A seventh aspect of the present invention relates to a method for treating articular cartilage in the sixth aspect, wherein the mesenchymal stem cells are derived from human synovium.

[0089] A method for treating articular cartilage according to an eighth aspect of the present invention is the method according to the sixth or seventh aspect, wherein the artificial tissue may further contain an extracellular matrix derived from the mesenchymal stem cells.

[0090] The mesenchymal stem cells according to aspect 9 of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are derived from synovium and form a scaffold-free artificial tissue having a three-dimensional structure cultured in a serum-free manner.

[0091] The mesenchymal stem cells according to aspect 10 of the present invention, in aspect 9, may be derived from human synovium.

[0092] The mesenchymal stem cells according to aspect 11 of the present invention, in accordance with aspect 9 or 10, may further contain an extracellular matrix derived from the mesenchymal stem cells.

[0093] The use of mesenchymal stem cells according to aspect 12 of the present invention is the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, wherein the mesenchymal stem cells are derived from synovium and form a scaffold-free artificial tissue having a three-dimensional structure cultured in a serum-free manner.

[0094] Aspect 13 of the present invention relates to the use of mesenchymal stem cells in aspect 12, wherein the mesenchymal stem cells are derived from human synovium.

[0095] Aspect 14 of the present invention relates to the use of mesenchymal stem cells in aspect 12 or 13, wherein the artificial tissue further contains an extracellular matrix derived from the mesenchymal stem cells. EXAMPLES

[0096] [Evaluation of cartilage regeneration of gMSC (registered trademark) 1] gMSC (registered trademark) 1 (manufactured by Two Cells Co., Ltd.) was used as a cell mass in which human synovium-derived mesenchymal stem cells were cultured in a serum-free environment to form a three-dimensional structure. The cartilage regeneration mechanism in which gMSC (registered trademark) 1 differentiates into cartilage was evaluated, and the equivalence and homogeneity of refrigerated gMSC (registered trademark) 1 and frozen gMSC (registered trademark) 1 were confirmed.

[0097] Ten 12-week-old nude rats were used as test animals. A defect was made in the rat knee cartilage over a range of 3-4 mm x 1.4 mm (rectangle) and 1 mm deep, and gMSC (registered trademark) 1 was transplanted and observed for 8 weeks. The following groups were used for evaluation: a non-application group (n=4) where no gMSC (registered trademark) 1 was applied, a group (n=8) where refrigerated gMSC (registered trademark) 1 was applied, and a group (n=8) where frozen gMSC (registered trademark) 1 was applied. Double staining of human vimentin and type II collagen (Col2) was performed at the defect site.

[0098] The cartilage regeneration function of gMSC (registered trademark) 1 was evaluated based on the degree of overlap between the human vimentin stained area and the Col2 stained area. The evaluation sites were three locations: the distal part of the defect, the proximal part, and the center. The degree of overlap between the human vimentin stained area and the Col2 stained area was blinded and scored. Scoring was performed according to the evaluation criteria shown in Figure 1. Figure 1 is a diagram showing the evaluation criteria for the cartilage regeneration function of an articular cartilage therapeutic agent.

[0099] Each of the three evaluation sites was scored on a three-level scale of 0 to 2 as shown in FIG. 1, and the score of the site with the highest value was taken as the score for that individual. The results are shown in FIG. 2. FIG. 2 is a diagram showing the evaluation results of the cartilage regeneration function of the articular cartilage therapeutic agent, expressed by the degree of overlap between the human vimentin stained area and the Col2 stained area. Compared with the non-application group, the refrigerated gMSC (registered trademark) 1 application group and the frozen gMSC (registered trademark) 1 application group had a high overlap score, and cartilage regeneration by the transplanted articular cartilage therapeutic agent was observed. In addition, from the 95% confidence interval of the difference between the refrigerated gMSC (registered trademark) 1 application group and the frozen gMSC (registered trademark) 1 application group and the non-application group, it was found that there was a similar degree of difference between each of the gMSC (registered trademark) 1 application groups and the non-application group.

[0100] In addition, histopathological examination was performed for each evaluation group using toluidine blue and SRY-box 9 (SOX9), and the results were compared with the results of double staining with human vimentin and Col2. Toluidine blue and SOX9 staining were evaluated on a three-level scale: -, ±, and +. The results are shown in Figure 3. Figure 3 shows the results of the histopathological examination. The images showing the results in Figure 3 also show the respective evaluation scores.

[0101] As shown in the area surrounded by the dashed line in the staining results for human vimentin and Col2 (Vin x Col2) in Figure 3, no human vimentin-positive area was confirmed in the non-application group, but a Col2-positive area was confirmed in the human vimentin-positive area in the refrigerated gMSC (registered trademark) 1 application group and the frozen gMSC (registered trademark) 1 application group. In addition, in individuals with a high score for the overlapping degree between the human vimentin staining area and the Col2 staining area, positivity for toluidine blue (TB) and SOX9 was also observed, confirming that the transplanted gMSC (registered trademark) 1 contributed to cartilage regeneration.

[0102] [Cartridge differentiation potential of gMSC (registered trademark) 1] Regarding the chondrocyte differentiation ability of gMSC1 itself, the expression of cartilage-related genes that are expressed in association with chondrocyte differentiation induction in vitro was confirmed by PCR measurement. gMSC1 (registered trademark) (manufactured by Two Cells Co., Ltd.) was stored under various storage conditions (no storage, refrigeration (10°C), freezing (-80°C)), and then pelleted (3D) cultured in chondrocyte differentiation induction medium. MB02-009, SYN098, and SYN107 were used as cell lines (n=3 each).

[0103] After extracting RNA from gMSCs (registered trademark) 1 after 1, 7, 14, and 21 days of differentiation induction and synthesizing cDNA, the time-dependent changes in gene expression under each storage condition were confirmed by qRT-PCR. The genes measured were as follows: COL1A2; COL2A1; COL10A1; ACAN; SOX9.

[0104] The results are shown in Figure 4. Figure 4 is a diagram showing the results of measuring the expression of cartilage-related genes. As shown in Figure 4, in all three cell lines, regardless of the storage conditions, increased expression of COL2A1, COL10A1, ACAN, and SOX9 was observed with the passage of the culture period, confirming that cartilage differentiation was induced. On the other hand, no increase in expression of COL1A2, an indicator of a fibrosis marker, was observed over time. This confirmed that gMSC (registered trademark) 1 has excellent cartilage differentiation ability regardless of the storage conditions.

[0105] [Ability of gMSC (registered trademark) 1 to secrete cartilage regeneration-related factors] The expression of cartilage regeneration-related factors secreted from transplanted gMSC (registered trademark) 1 was confirmed in vitro by ELISA measurement. gMSC (registered trademark) 1 (manufactured by Two Cells Co., Ltd.) was stored under each storage condition (no storage, refrigeration (10°C), freezing (-80°C)) and then incubated in culture medium. MB02-009, SYN098, and SYN107 were used as cell lines (n=3 each). After 2 days, the culture supernatant and protein extract extracted from gMSC (registered trademark) 1 were collected, and the expression level of each factor was confirmed by ELISA. The measured factors were as follows: TGF-b1; Fibronectin; TIMP-1; TSP-2; FGF-2.

[0106] The results are shown in Figures 5 and 6. Figure 5 shows the results of measuring the expression levels of cartilage regeneration-related factors in the culture supernatant, and Figure 6 shows the results of measuring the expression levels of cartilage regeneration-related factors in gMSC (registered trademark) 1 extract. As shown in Figures 4 and 5, both the culture supernatant and the gMSC (registered trademark) 1 extract showed significant differences in expression levels compared to the control STK2 medium and extraction reagent. No clear differences due to cell lines and storage conditions were confirmed. This suggests that gMSC (registered trademark) 1 may secrete cartilage regeneration-related factors after transplantation and regenerate defective cartilage by autocrine or paracrine action.

[0107] [Additional Notes] The present invention is not limited to the above-described embodiments or examples, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in the different embodiments or examples are also included in the technical scope of the present invention. [Industrial Applicability]

[0108] The present invention can provide a highly useful transplantation therapy using mesenchymal stem cells, and can therefore be suitably used in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. An articular cartilage treatment agent comprising a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured in a serum-free environment form a three-dimensional structure.

2. The articular cartilage therapeutic agent according to claim 1 , wherein the mesenchymal stem cells are derived from human synovium.

3. The articular cartilage therapeutic agent according to claim 1 or 2, further comprising an extracellular matrix derived from said mesenchymal stem cells.

4. The articular cartilage treating agent according to claim 1 or 2, further comprising a composition for cryopreservation comprising at least one component selected from the group consisting of fatty acids and fatty acid esters.

5. A step of detecting the presence of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage therapeutic agent according to claim 1 or 2 has been administered. The method for evaluating an agent for treating articular cartilage comprises the steps of:

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