Method for treating articular cartilage

Abstract

Provided is a highly safe method for treating articular cartilage. This method for treating articular cartilage comprises a step for introducing a scaffold-free artificial tissue in which serum-free cultured synovial membrane-derived mesenchymal stem cells form a three-dimensional structure.

Description

Treatment methods for articular cartilage

[0001] The present invention relates to a method for treating articular cartilage.

[0002] Articular cartilage plays a vital role in smooth joint movement and shock absorption during weight-bearing. Damage to articular cartilage can lead to movement disorders and pain, significantly impacting daily life. In recent years, regenerative medicine using cell transplantation has been applied to treat such articular cartilage damage.

[0003] As a cell applicable to regenerative medicine, for example, Patent Document 1 proposes a scaffold-free, self-assembling, three-dimensional artificial tissue having tissue strength suitable for clinical application.

[0004] International Publication No. 2005 / 012512 brochure

[0005] Cells used in regenerative medicine require a high level of safety and quality, and there is room for improvement in the artificial tissue described in Patent Document 1. One aspect of the present invention aims to provide a highly safe articular cartilage treatment agent.

[0006] To solve the above problems, an articular cartilage treatment agent according to one aspect of the present invention contains a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells have formed a three-dimensional structure.

[0007] According to one aspect of the present invention, a highly safe articular cartilage treatment agent can be provided.

[0008] This figure shows the evaluation criteria for the cartilage regeneration function of the articular cartilage treatment agent used in the examples. This figure shows the evaluation results of the cartilage regeneration function of the articular cartilage treatment agent in the examples. This figure shows the histopathological examination results in the examples. This figure shows the results of measuring cartilage-related gene expression in the examples. This figure shows the results of measuring the expression levels of cartilage regeneration-related factors in the culture supernatant in the examples. This figure shows the results of measuring the expression levels of cartilage regeneration-related factors in gMSC® 1 extract in the examples.

[0009] Embodiments of the present invention are described below, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A to B" indicating a numerical range means "A or greater, and B or less".

[0010] [Articular cartilage treatment agent] An articular cartilage treatment agent according to one aspect of the present invention contains a scaffold-free artificial tissue in which serum-free cultured synovial membrane-derived mesenchymal stem cells have formed a three-dimensional structure.

[0011] The inventors have discovered that a scaffold-free artificial tissue formed from serum-free cultured synovial mesenchymal stem cells that have formed a three-dimensional structure is effective in treating articular cartilage. Furthermore, the inventors have found that this scaffold-free artificial tissue formed from serum-free cultured synovial mesenchymal stem cells that have formed a three-dimensional structure engrafts well at the transplant site and differentiates favorably into chondrocytes. As a result, an articular cartilage treatment agent containing a scaffold-free artificial tissue formed from serum-free cultured synovial mesenchymal stem cells that have formed a three-dimensional structure exhibits superior therapeutic effects.

[0012] Articular cartilage treatment agents can be used to treat damaged or missing articular cartilage. By administering the agent to the area requiring treatment, it treats the damage or loss of articular cartilage. Administering the agent to the damaged or missing articular cartilage site promotes regeneration of the articular cartilage at the administration site, thereby treating the damage or loss. Articular cartilage treatment agents can be used to treat cartilage in joints at any location, such as the knee, elbow, and shoulder joints. The articular cartilage treated by the articular cartilage treatment agent may include meniscal tissue.

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

[0014] (Mesenchymal stem cells derived from synovial membrane) In this specification, "mesenchymal stem cells" refers to somatic stem cells that differentiate into tissues belonging to the mesenchyme. Mesenchymal stem cells also include those isolated from mesenchymal stem cells that have specific properties, those that have been stimulated in some way, such as by cytokine stimulation, and those that have been genetically modified. For example, MUSE cells, MACC cells, and SP-1 cells are also included in mesenchymal stem cells. Mesenchymal stem cells have proliferative capacity and the ability to differentiate into osteocytes, 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, synovial membrane, alveolar bone, and periodontal ligament, but also from the placenta, umbilical cord, umbilical cord blood, and various fetal tissues.

[0015] The synovial-derived mesenchymal stem cells that form the artificial tissue contained in articular cartilage treatment agents are stem cells contained in the synovial membrane and are obtained from synovial tissue by known methods. Synovial-derived mesenchymal stem cells have the ability to differentiate into chondrocytes. Synovial-derived mesenchymal stem cells may be synovial-derived mesenchymal stem cells from non-human animals such as rats and mice, but it is preferable that they be human synovial-derived mesenchymal stem cells. Human synovial-derived mesenchymal stem cells have better engraftment at the treatment site and superior therapeutic effect when the articular cartilage treatment agent is administered to the treatment site in humans. In addition, human synovial-derived mesenchymal stem cells can reduce the risk of biological contamination and the presence of immunogenic substances in humans receiving the articular cartilage treatment agent.

[0016] Synovial membrane-derived mesenchymal stem cells are cultured in a serum-free culture. With serum-free culture, the culture components are known. That is, because serum is derived from natural components, there are differences in components from batch to batch, but such differences do not occur in serum-free culture media. Therefore, serum-free cultured synovial membrane-derived mesenchymal stem cells are superior in both safety and quality. Furthermore, the risks of biological contamination and the presence of immunogenic substances are minimized, and the presence of substances not present in the human body is minimized. Also, since the contained biological raw materials are clearly defined, quality control is easy. In addition, mesenchymal stem cells cultured in certain serum-free media, such as STK® medium, exhibit superior proliferation rates.

[0017] In this specification, "serum-free culture" is intended to mean a culture that does not use serum. For example, it is intended to mean a culture using serum-free medium, which is a culture medium that does not contain serum.

[0018] (Serum-free culture) An example of a serum-free culture medium used for serum-free culture of synovial-derived mesenchymal stem cells that form artificial tissues contained in articular cartilage treatment agents is described below. The basal medium for constituting the serum-free medium is not particularly limited as long as it is an animal cell medium 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 individually 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 1:1 ratio.

[0019] In one embodiment, a serum-free medium prepared 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 synovial mesenchymal stem cells. The FGF content in the basal medium is preferably 0.1 to 100 ng / ml at a final concentration, and more preferably 3 ng / ml. The PDGF content in the basal medium is preferably 0.5 to 100 ng / ml at a final concentration, and more preferably 10 ng / ml. The TGF-β content in the basal medium is preferably 0.5 to 100 ng / ml at a final concentration, and more preferably 10 ng / ml.

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

[0021] By using such serum-free media, it is possible to prevent contamination by foreign proteins while achieving a proliferation-promoting effect equivalent to or better than that of serum-containing media, thereby allowing synovial-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 individually or in combination. In one embodiment, the serum-free medium may contain a combination of phosphatidic acid and phosphatidylcholine, and these phospholipids may be of animal or plant origin.

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

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

[0025] As used herein, HGF refers to a growth factor selected from the hepatocyte growth factor family, and EGF refers to 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 ascorbic acid compounds.

[0027] As used herein, ascorbic acid compounds refer to ascorbic acid (vitamin C), ascorbic acid diphosphate, or compounds similar thereto.

[0028] Furthermore, the growth factors contained in the serum-free culture medium may be naturally occurring or manufactured through genetic engineering.

[0029] In one aspect, the serum-free medium preferably contains a lipid antioxidant. In one embodiment, the lipid antioxidant contained in the serum-free medium may be DL-α-tocopherol acetate (vitamin E). The serum-free medium may also 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 serenate. Where used herein, insulin may be insulin-like growth factor, and may be derived from natural cells or produced by genetic engineering. A culture medium additive according to one aspect 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 synovial tissue of humans or other animals by conventionally known methods are seeded in the serum-free medium described above and cultured until they proliferate to the desired number. The culture conditions are 1 to 2 × 10⁶ cells per 1 ml of medium. ４It is preferable to seed individual mesenchymal stem cells, the culture temperature is 37°C ± 1°C, the culture time is 48 to 96 hours, and it is preferable to culture under 5% CO ２ 2. By culturing in this way, mesenchymal stem cells with maintained or improved immunosuppressive ability can be efficiently obtained in large quantities.

[0032] The culture vessel used for culturing is not particularly limited as long as mesenchymal stem cells can proliferate. For example, a 75 cm Falcon ２ flask, a 75 cm Sumitomo Bakelite ２ flask, etc. can be preferably used. However, depending on the cells, cell proliferation may be affected by the type of culture vessel used. Therefore, in order to more efficiently proliferate synovium-derived mesenchymal stem cells, it is preferable to culture using a culture vessel suitable for proliferation for each mesenchymal stem cell to be proliferated (hereinafter, also referred to as "proliferation target cell").

[0033] As a method for selecting a culture vessel suitable for the proliferation of proliferation target cells, for example, a method of allowing the proliferation target cells to select the optimal culture vessel can be cited. Specifically, a plurality of types of culture vessels are prepared, and the proliferation target cells are proliferated under the same culture conditions except that the types of culture vessels are different. The number of cells two weeks after the start of culture is measured by a known method, and it can be determined that the culture vessel is suitable for the proliferation of the proliferation target cells in order from the one with the largest number of cells. Also, when the proliferation rate of the proliferation target cells is high, even before two weeks have passed since the start of culture, it can be determined that the culture vessel is suitable for the proliferation of the proliferation target cells in order from the one with the shortest period to reach 80 to 90% of the cell number in the confluent state.

[0034] In addition, for the proliferation of mesenchymal stem cells, it is an essential condition for the cells to adhere to the culture vessel. Therefore, when the adhesion of the proliferation target cells to the culture vessel is weak, it is preferable to further contain a cell adhesion molecule in the serum-free medium when culturing in a serum-free manner. Examples of cell adhesion molecules include fibronectin, collagen, gelatin, etc. These cell adhesion molecules may be used alone or in combination of multiple types.

[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 the final concentration. In one embodiment, when fibronectin is used as the cell adhesion molecule, by adding it so that the final concentration of fibronectin in the serum-free medium becomes 5 μg / ml, the adhesion efficiency of the target cells for proliferation to the culture vessel can be improved.

[0036] In addition, in serum-free culture, the synovium-derived mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells proliferate in a scaffold-dependent manner, when the mesenchymal stem cells are locally biased and proliferating, the culture conditions can be improved by passaging the synovium-derived mesenchymal stem cells during proliferation.

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

[0038] (Artificial tissue) The artificial tissue contained in the articular cartilage therapeutic agent according to one aspect 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 can be a tissue-engineered construct (TEC). When cells are transplanted by administering a cell suspension to the affected area, there are reports that the administered cells are likely to detach from the transplantation site and do not remain at the transplantation site. However, since the articular cartilage therapeutic agent according to one aspect of the present invention contains a scaffold-free artificial tissue having a three-dimensional structure, the cells are likely to engraft at the transplantation site.

[0039] In addition, for the scaffold-free artificial tissue that forms a three-dimensional structure, a cell mass of mesenchymal stem cells processed into a three-dimensional structure by a conventionally known method may be used. In this specification, when referring to an "artificial tissue" as having a "three-dimensional structure", it refers to an object that extends in a three-dimensional direction including cells in which the matrix is three-dimensionally oriented, the cells are arranged three-dimensionally, and the cells maintain cell-cell binding and orientation.

[0040] As the artificial tissue, an area, thickness, and strength suitable for the treatment of articular cartilage may be appropriately set, and those skilled in the art can appropriately set its size. This size can be set according to the transplantation environment. Small-sized artificial tissues have the advantage that they can be injected into the body cavity with an injection needle. Also, large-sized artificial tissues have the advantage that it is easy to administer a sufficient number of cells because, for example, they are easy to handle, such as being easy to grip with forceps during surgery.

[0041] When the artificial tissue is transplanted, it preferably has at least a certain size. Such a size is, for example, 1 cm ２ or more for the area of the artificial tissue that forms a three-dimensional structure, preferably 2 cm ２ or more, more preferably 3 cm ２ or more. Even more preferably 4 cm ２ or more, 5 cm ２ or more, 6 cm ２ or more, 7 cm ２ or more, 8 cm ２ or more, 9 cm ２ or more, 10 cm ２ or more, 15 cm ２ or more, or 20 cm ２ or more. Also, for example, 40 cm ２ or less, 30 cm ２ or less, 20 cm ２ or less, but is not limited thereto, and the area can be 1 cm ２ or less, or 40 cm ２ or more depending on the application.

[0042] When expressed in terms of artificial tissue volume, the above size is preferably 2 mm. ３ The above is more preferable to 40 mm ３ That's all, and also, for example, 40 cm ３ Below, or 20 cm ３ 2 mm ３ The following are also possible.

[0043] The sufficient thickness of the implantable artificial tissue varies depending on the area to be implanted, but those skilled in the art can set the thickness appropriately. This thickness can be set according to the environment in which it is implanted. The thickness of the artificial tissue is intended to be 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 any case, it may also be 1 mm or less, 10 mm or less, or 5 mm or less.

[0044] The number of cells that make up the artificial tissue can be selected as appropriate; for example, it may be a cluster of 50 to 200 cells, or a cluster of 1 million to 100 million cells. Furthermore, the cluster may be small or large.

[0045] (Scaffold-Free) In this specification, "scaffold-free" means substantially free from materials (scaffolds) conventionally used in the production of artificial tissues. Examples of such scaffold materials include, but are not limited to, chemical polymers, ceramics, or biologics such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold is a material that is substantially solid and has sufficient strength to support cells or tissues.

[0046] Traditionally, the mainstream cell preparations have been "scaffold-type" cell preparations, which are processed into three-dimensional structures by artificially adding scaffolds—materials that serve as a scaffold for cells and tissues to attach to or hold them and enable their growth. However, recently, due to concerns about the risks of artificially adding materials, development is progressing on "scaffold-free" cell preparations, which are manufactured without artificially adding scaffolds. These are produced by methods such as stimulating the cells themselves to produce an environment that serves as their own scaffold.

[0047] Because the artificial tissue is a scaffold-free three-dimensional structure, the amount of materials other than mesenchymal stem cells contained in articular cartilage treatment agents can be reduced. Furthermore, because 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 are minimized, and the amount of substances not present in the body is 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, because the components are known due to the scaffold-free three-dimensional structure, safety and quality stability are superior. In addition, the aforementioned natural products carry the risk of biological contamination and the presence of immunogenic substances. By using a scaffold-free three-dimensional structure for the artificial tissue, these 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 synovial-derived mesenchymal stem cells, contained in an articular cartilage treatment agent according to one aspect of the present invention, for example, a conventionally known method using a low-adhesion plate, a micropatterned surface plate, etc., and a hanging drop method can be employed. Alternatively, the artificial tissue may be prepared using the method described in Japanese Patent No. 4522994. Commercially available products may also be used, for example, gMSC® 1 (manufactured by TwoCell Co., Ltd.) can be suitably used.

[0049] (Extracellular Matrix) An articular cartilage therapeutic agent according to one aspect of the present invention may further contain an extracellular matrix derived from mesenchymal stem cells. In this specification, "extracellular matrix" also refers to the extracellular matrix, which is a substance present between somatic cells, whether epithelial or non-epithelial cells.

[0050] The extracellular matrix is ​​one of the biomolecules produced by cells and is known to be involved not only in supporting tissues but also in creating the internal environment necessary for the survival of all somatic cells. Typical extracellular matrix components include, for example, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, and proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), but is not limited to these. Various extracellular matrix components that play a role in cell adhesion can be used in this 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 it may exist independently of the artificial tissue within the articular cartilage therapeutic agent. The articular cartilage therapeutic agent may further include reagents, buffers, etc., for stably maintaining the artificial tissue.

[0052] (Cryopreservation Composition) An articular cartilage treatment agent according to one aspect of the present invention may further include a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters. In this specification, "cryopreservation composition" is a cryopreservation composition for cryopreserving scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells have formed a three-dimensional structure. The articular cartilage treatment agent can be cryopreserved by further including the cryopreservation composition, and is frozen well with good recovery and survival rates, and the recovery and survival rates after cryopreservation and thawing are also good. Furthermore, the quality of the artificial tissue after thawing of the cryopreserved articular cartilage treatment agent is normal, and the properties of the artificial tissue do not change even after cryopreservation and thawing. Therefore, it can be cryopreserved until needed and used when required.

[0053] Conventional cell therapies are transported and stored in non-freezing conditions, such as refrigeration, but these cell therapies have a short shelf life of only a few days after manufacturing. As a result, quality control tests, including safety tests such as sterility tests, which require several days to obtain results, may not be adequately carried out before shipment and transplantation. According to one aspect of the present invention, cryopreservation makes it possible to prevent changes in the quality of articular cartilage therapy agents for, for example, several months. Therefore, it is possible to prevent changes in the quality of articular cartilage therapy agents during the quality control testing period, and to ship and transplant them after their safety has been sufficiently confirmed.

[0054] Examples of fatty acids included in cryopreservation compositions include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Among these, it is preferable that at least one of linoleic acid and linolenic acid is included in the cryopreservation composition. The inclusion of at least one of linoleic acid and linolenic acid in the cryopreservation composition allows for the cryopreservation of mesenchymal stem cells with a higher survival rate. Short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids are also acceptable. Furthermore, highly unsaturated fatty acids are also acceptable. These may be used individually or as a mixture of multiple types. Among these, a mixture of multiple fatty acids is preferred, and a mixture in which the types and amounts of fatty acids are specified is more preferable. For example, Chemically defined lipid concentrate (Thermo Fisher Scientific Inc., catalog number 11905-031) is more preferred.

[0055] As mentioned above, the fatty acids exemplified above may be used individually or in combination, but it is more preferable to use a mixture of multiple types. Including a mixture of more types of fatty acids in the cryopreservation composition allows for better survival rates when freezing artificial tissues, and prevents changes in the properties of the artificial tissues after thawing.

[0056] The fatty acid content in the cryopreservation composition is not particularly limited, but it is preferably, for example, 0.01 μg / ml to 500 μg / ml at the final concentration relative to the total amount of the cryopreservation composition. For example, 1 / 1000 to 1 / 10 (v / v) relative to the total amount of the cryopreservation composition is more preferable.

[0057] Examples of fatty acid esters included in cryopreservation compositions include phospholipids and triglycerides. Among these, it is preferable that phospholipids be included in the cryopreservation composition. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.

[0058] When using fatty acid esters such as phospholipids, it is preferable that their content be such that the final concentration in the cryopreserved composition at the time of use is 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreserved composition.

[0059] The cryopreservation composition may further contain a substance that assists in the water solubility (emulsification) of fatty acids or fatty acid esters, such as a surfactant. The surfactant may be Pluronic F-68 or Tween 80.

[0060] The components of the cryopreservation composition may include a cryoprotective agent that inhibits the growth of ice crystals within cells during the freezing and thawing process. Examples of cryoprotective agents include DMSO (dimethyl sulfoxide). When the cryopreservation composition contains a cryoprotective agent, the amount is more preferably 0.5% to 50% (v / v) of the total amount of the cryopreservation composition.

[0061] The cryopreservation composition may further contain other components, such as a base culture medium, a thickener, a pH adjuster, or a cryoprotectant.

[0062] Furthermore, the cryopreservation composition is more preferably to contain insulin, albumin, and transferrin. Insulin, albumin, and transferrin can enhance the effects of fatty acids. When these are added, it is preferable that the final concentration in the cryopreservation composition at the time of use be between 0.5 μg / ml and 500 μg / ml.

[0063] Articular cartilage treatment agents can be cryopreserved by a method comprising the steps of immersing a scaffold-free artificial tissue, in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure, in a solution of a cryopreservation composition, and freezing the artificial tissue in the solution. The freezing temperature can be appropriately set to the temperature at which the artificial tissue to be frozen freezes, for example, -80°C or below, or -196°C or below. When freezing at -80°C or below, for example, conventionally known methods can be used, and when freezing at -196°C or below, liquid nitrogen can be used.

[0064] Cryopreserved articular cartilage treatment agents can be used after thawing at a temperature that does not damage the scaffold-free artificial tissue, which is formed from serum-free cultured synovial-derived mesenchymal stem cells that have created a three-dimensional structure. Common thawing methods include, for example, thawing by water bath, heat block, and room temperature thawing.

[0065] The melting temperature is preferably 10°C or higher and 45°C or lower, more preferably 20°C or higher and 40°C or lower. For example, it may be left standing at room temperature (25°C), but it is more preferable to melt it using a water bath at 35-38°C, as shown in the examples.

[0066] [Method for Evaluating Articular Cartilage Therapeutic Agents] An evaluation method for articular cartilage therapeutic agents according to one aspect of the present invention includes a 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 aspect of the present invention is administered. According to the evaluation method for articular cartilage therapeutic agents, the therapeutic effect of the articular cartilage therapeutic agent on articular cartilage is evaluated using the differentiation of mesenchymal stem cells contained in the articular cartilage therapeutic agent into articular chondrocytes as an indicator at the site where the articular cartilage therapeutic agent is administered.

[0067] In the evaluation process, by detecting the presence of a mixture of mesenchymal stem cells and articular chondrocytes at the site where the articular cartilage treatment 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 whether the articular cartilage treatment agent functions in treating the joint.

[0068] The presence of a mixture of mesenchymal stem cells and articular chondrocytes at the site where an articular cartilage treatment 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 articular chondrocytes for type II collagen. Staining of human vimentin and type II collagen can be performed by conventionally known methods.

[0069] In the evaluation process, if double staining reveals that only stained human vimentin is detected at the site where the articular cartilage treatment agent was administered, it can be determined that mesenchymal stem cells and articular chondrocytes are not mixed. As a result, it can be concluded that the administered mesenchymal stem cells did not differentiate into articular chondrocytes, and that the articular cartilage treatment agent is not functioning to treat the joint.

[0070] Furthermore, if both stained human vimentin and type II collagen are detected at the site where the articular cartilage treatment agent was administered as a result of double staining, it can be evaluated that the administered mesenchymal stem cells have differentiated into articular chondrocytes and that the articular cartilage treatment agent is functioning in joint treatment.

[0071] In the evaluation process, it is preferable to detect the presence of stained type II collagen within the stained human vimentin region. By detecting the overlap between the human vimentin region and the type II collagen region in this way, it is possible to more reliably evaluate that the administered mesenchymal stem cells have differentiated into articular chondrocytes. Furthermore, if type II collagen in such overlapping regions is stained to the same intensity as that of normal articular chondrocytes, it can be evaluated that the joint therapeutic function of the articular cartilage treatment agent is superior.

[0072] The evaluation method for articular cartilage treatment agents allows for accurate assessment of articular cartilage regeneration by the administered agent. This method also allows for the differentiation and evaluation of spontaneous healing at the site of articular cartilage damage or defect from treatment with articular cartilage treatment agents. As a result, in addition to evaluating the therapeutic effect of articular cartilage treatment agents, the mechanism of articular cartilage regeneration by these agents can be assessed.

[0073] The evaluation method for articular cartilage treatment agents may target animals including humans, but may also target animals other than humans. Among animals other than humans, for example, non-human mammals, it is possible to evaluate the therapeutic effect of the articular cartilage treatment agent on humans to some extent from the evaluation results on these animals.

[0074] [Method for treating articular cartilage] A method for treating articular cartilage according to one aspect of the present invention includes the step of administering a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells have formed a three-dimensional structure. Furthermore, the mesenchymal stem cells that form the artificial tissue administered in the method for treating articular cartilage may be derived from human synovial membrane. In addition, the artificial tissue administered in the method for treating articular cartilage may further contain an extracellular matrix derived from mesenchymal stem cells. That is, 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, so the explanation of the artificial tissue will be based on the explanation of the artificial tissue in the articular cartilage therapeutic agent.

[0075] In the administration process, the artificial tissue can be administered by transplanting it to the treatment site or by injecting an injection solution containing the artificial tissue into the treatment site. In the administration process, the artificial tissue may be administered alone or in combination with other therapeutic agents.

[0076] The amount of artificial tissue administered during the administration process can be appropriately determined by a person 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 morphology or type of the tissue. The frequency of administration of the artificial tissue during the administration process can also be appropriately determined by a person 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 administration frequencies include daily administration to once every few months (for example, once a week to once a month). The amount and frequency of administration of the artificial tissue during the administration process may be appropriately adjusted according to the course of treatment.

[0077] In the administration process, the treatment site to which the artificial tissue is administered is the site of damage or loss of articular cartilage. By administering artificial tissue to the site of damage or loss of articular cartilage, regeneration of articular cartilage is promoted, and the damage or loss of articular cartilage can be treated.

[0078] [Mesenchymal Stem Cells for Use in the Treatment of Articular Cartilage] Mesenchymal stem cells according to one aspect of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are synovial-derived, serum-free cultured, and form a three-dimensional, scaffold-free artificial tissue. Furthermore, mesenchymal stem cells for use in the treatment of articular cartilage may be derived from human synovial membrane. In addition, 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 mesenchymal stem cells for use in the treatment of articular cartilage are mesenchymal stem cells that form an artificial tissue contained in an articular cartilage therapeutic agent according to one aspect of the present invention, the explanation of mesenchymal stem cells will be based on the explanation of mesenchymal stem cells and artificial tissue in articular cartilage therapeutic agents.

[0079] Mesenchymal stem cells for use in the treatment of articular cartilage can be used to treat damaged or defective areas of articular cartilage. By administering mesenchymal stem cells for use in the treatment of articular cartilage to the site requiring treatment, damage or defects in articular cartilage can be treated. Administering mesenchymal stem cells for use in the treatment of articular cartilage to the damaged or defective area of ​​articular cartilage promotes regeneration of articular cartilage at the administration site, thereby treating the damage or defect.

[0080] [Use of Mesenchymal Stem Cells for Manufacturing Articular Cartilage Therapeutic Agents] The use of mesenchymal stem cells for manufacturing articular cartilage therapy agents according to one aspect of the present invention is the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, wherein the mesenchymal stem cells are synovial-derived, serum-free cultured, and form a three-dimensional, scaffold-free artificial tissue. Furthermore, in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, the mesenchymal stem cells may be derived from human synovial membrane. Moreover, in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, the artificial tissue may further contain extracellular matrix derived from mesenchymal stem cells. That is, one aspect of the artificial tissue in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents is the artificial tissue contained in articular cartilage therapy agents according to one aspect of the present invention, so the explanation of the artificial tissue will be based on the explanation of the artificial tissue in articular cartilage therapy agents.

[0081] Articular cartilage therapeutic agents manufactured using mesenchymal stem cells may further include pharmaceutically acceptable carriers, along with a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure. Pharmaceutically acceptable carriers included in articular cartilage therapeutic agents include any substance known in the field. Examples of pharmaceutically acceptable carriers include, but are not limited to, antioxidants, preservatives, colorants, flavorings, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, excipients, and / or pharmaceutical adjuvants.

[0082] Articular cartilage therapeutic agents manufactured using mesenchymal stem cells can be used to treat damaged or defective articular cartilage. These agents treat articular cartilage damage or defects by being administered to the area requiring treatment. By administering the articular cartilage therapeutic agent to the damaged or defective articular cartilage site, regeneration of articular cartilage at the administration site is promoted, thereby treating the damage or defect.

[0083] [Summary] The articular cartilage treatment agent according to Embodiment 1 of the present invention contains a scaffold-free artificial tissue in which serum-free cultured synovial membrane-derived mesenchymal stem cells have formed a three-dimensional structure.

[0084] In the articular cartilage treatment agent according to embodiment 2 of the present invention, the mesenchymal stem cells may be derived from human synovial membrane in embodiment 1.

[0085] The articular cartilage treatment agent according to embodiment 3 of the present invention may further contain the extracellular matrix derived from the mesenchymal stem cells in embodiment 1 or 2.

[0086] The articular cartilage treatment agent according to embodiment 4 of the present invention may further include a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters, and may be cryopreserved, in any of embodiments 1 to 3.

[0087] A method for evaluating an articular cartilage therapeutic agent according to aspect 5 of the present invention includes a step of detecting the presence of the mesenchymal stem cells and articular cartilage cells at the site where the articular cartilage therapeutic agent according to any of aspects 1 to 4 is administered.

[0088] A method for treating articular cartilage according to aspect 6 of the present invention includes the step of administering a scaffold-free artificial tissue in which mesenchymal stem cells derived from synovium, cultured without serum, have formed a three-dimensional structure.

[0089] In the method for treating articular cartilage according to embodiment 7 of the present invention, in embodiment 6, the mesenchymal stem cells may be derived from human synovial membrane.

[0090] In the method for treating articular cartilage according to aspect 8 of the present invention, in aspect 6 or 7, the artificial tissue may further contain the extracellular matrix derived from the mesenchymal stem cells.

[0091] 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 synovial-derived, serum-free cultured, and form a scaffold-free artificial tissue that is a three-dimensional structure.

[0092] The mesenchymal stem cells according to embodiment 10 of the present invention may be derived from human synovial membrane in embodiment 9.

[0093] The mesenchymal stem cells according to embodiment 11 of the present invention may further contain an extracellular matrix derived from the mesenchymal stem cells in embodiment 9 or 10.

[0094] The use of mesenchymal stem cells according to aspect 12 of the present invention is the use of mesenchymal stem cells for the production of an articular cartilage therapeutic agent, wherein the mesenchymal stem cells are synovial-derived and form a scaffold-free artificial tissue that is a three-dimensional structure cultured without serum.

[0095] The use of mesenchymal stem cells according to embodiment 13 of the present invention is such that, in embodiment 12, the mesenchymal stem cells may be derived from human synovial membrane.

[0096] The use of mesenchymal stem cells according to embodiment 14 of the present invention is such that, in embodiment 12 or 13, the artificial tissue further contains an extracellular matrix derived from the mesenchymal stem cells.

[0097] A method for treating articular cartilage according to embodiment 15 of the present invention further comprises, in any of embodiments 6 to 8, a cryopreservation composition in which the artificial tissue further contains at least one component selected from the group consisting of fatty acids and fatty acid esters, a step of thawing the cryopreserved artificial tissue, and in the administration step, the thawed artificial tissue may be administered.

[0098] [Evaluation of cartilage regeneration of gMSC® 1] gMSC® 1 (manufactured by TwoCell Co., Ltd.) was used as a cell aggregate formed by serum-free cultured human synovial membrane-derived mesenchymal stem cells forming a three-dimensional structure. The cartilage regeneration mechanism by which gMSC® 1 differentiates into cartilage was evaluated, and the equivalence and homogeneity of refrigerated gMSC® 1 and frozen gMSC® 1 were confirmed.

[0099] Ten 12-week-old nude rats were used as test animals. A 3-4 mm x 1.4 mm (rectangular) area with a depth of 1 mm was created in the knee cartilage of the rats, and gMSC® 1 was transplanted. The rats were observed for 8 weeks. Three groups were used for evaluation: a control group (n=4) without gMSC® 1, a group with refrigerated gMSC® 1 (n=8), and a group with frozen gMSC® 1 (n=8). Double staining for human vimentin and type II collagen (Col2) was performed at the defect site.

[0100] The cartilage regeneration function of gMSC® 1 was evaluated based on the degree of overlap between the human vimentin-stained area and the Col2-stained area. The evaluation sites were the distal, proximal, and central parts of the defect. 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 articular cartilage treatment agents.

[0101] Each of the three evaluation sites was scored on a three-point scale from 0 to 2, as shown in Figure 1, and the score of the site with the highest numerical value was taken as the score for that individual. The results are shown in Figure 2. Figure 2 shows the evaluation results of the cartilage regeneration function of the articular cartilage treatment agent, represented by the degree of overlap between the human vimentin-stained area and the Col2-stained area. Compared to the no-treatment group, the refrigerated gMSC® 1 treatment group and the frozen gMSC® 1 treatment group had higher overlap scores, indicating cartilage regeneration by the transplanted articular cartilage treatment agent. Furthermore, from the 95% confidence interval of the difference between the refrigerated gMSC® 1 treatment group and the frozen gMSC® 1 treatment group and the no-treatment group, it was found that there was a similar degree of difference between each gMSC® 1 treatment group and the no-treatment group.

[0102] Furthermore, histopathological examinations using toluidine blue and SRY-box 9 (SOX9) were performed on each evaluation group and compared with the results of double staining for human vimentin and Col2. Toluidine blue and SOX9 staining were evaluated on a three-point scale: -, ±, and +. The results are shown in Figure 3. Figure 3 shows the results of the histopathological examinations. The evaluation scores for each result are also shown in the images in Figure 3.

[0103] In Figure 3, as shown in the dashed areas in the staining results for human vimentin and Col2 (Vin × Col2), no human vimentin-positive areas were observed in the untreated group. However, in the refrigerated gMSC® 1 treated group and the frozen gMSC® 1 treated group, Col2-positive areas were observed within the human vimentin-positive areas. Furthermore, individuals with a high score for the degree of overlap between the human vimentin-stained areas and the Col2-stained areas also showed positivity for toluidine blue (TB) and SOX9, confirming that the transplanted gMSC® 1 contributed to cartilage regeneration.

[0104] [Chondrosis Differentiation Potential of gMSC® 1] Regarding the chondrosis differentiation potential of gMSC® 1 itself, the expression of cartilage-related genes expressed in conjunction with in vitro chondrosis induction was confirmed by PCR measurement. gMSC® 1 (manufactured by TwoCell Co., Ltd.) was stored under various storage conditions (no storage, refrigeration (10°C), freezing (-80°C)) and then cultured as pellets (3D) in chondrosis differentiation induction medium. MB02-009, SYN098, and SYN107 were used as cell lines (n=3 each).

[0105] RNA was extracted from gMSC(registered trademark) 1 at differentiation induction days 1, 7, 14, and 21, and cDNA synthesis was performed. After that, the changes in gene expression over time under each storage condition were confirmed by qRT-PCR. The genes measured were as follows: COL1A2; COL2A1; COL10A1; ACAN; SOX9.

[0106] The results are shown in Figure 4. Figure 4 shows the results of measuring cartilage-related gene expression. As shown in Figure 4, in all three cell lines, regardless of storage conditions, an increase in the expression of COL2A1, COL10A1, ACAN, and SOX9 was observed with the progression of the culture period, confirming that cartilage differentiation was induced. On the other hand, no increase in the expression of COL1A2, an indicator of fibrosis, was observed over time. From this, it was confirmed that gMSC(registered trademark) 1 has excellent cartilage differentiation ability regardless of storage conditions.

[0107] [Secretion capacity of cartilage regeneration-related factors of gMSC® 1] The expression of cartilage regeneration-related factors secreted from transplanted gMSC® 1 was confirmed in vitro by ELISA measurement. gMSC® 1 (manufactured by TwoCell Co., Ltd.) was stored under various storage conditions (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 extracts extracted from gMSC® 1 were collected, and the expression levels of each factor were confirmed by ELISA. The measured factors were as follows: TGF-β1; Fibronectin; TIMP-1; TSP-2; FGF-2.

[0108] 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 the gMSC® 1 extract. As shown in Figures 5 and 6, significant differences in expression levels were observed in both the culture supernatant and the gMSC® 1 extract compared to the control STK2 medium and extraction reagent. No clear differences were observed due to cell line or storage conditions. This suggests that gMSC® 1 may secrete cartilage regeneration-related factors after transplantation and regenerate defective cartilage through autocrine or paracrine action.

[0109] [Additional Notes] The present invention is not limited to the embodiments or examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention.

[0110] The present invention provides a highly valuable transplantation therapy agent using mesenchymal stem cells, making it suitable for use in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. A method for treating articular cartilage, comprising the step of administering a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure.

2. The method for treating articular cartilage according to claim 1, wherein the mesenchymal stem cells are derived from human synovial membrane.

3. The method for treating articular cartilage according to claim 1, wherein the artificial tissue further contains the extracellular matrix derived from the mesenchymal stem cells.

4. The method for treating articular cartilage according to claim 1, further comprising the steps of: the artificial tissue further contains a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters; the method further comprising the step of thawing the cryopreserved artificial tissue; and the administration step comprising administering the thawed artificial tissue.

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