Method for producing cell preparations for joint therapy, cell preparations for joint therapy, and method for culturing mesenchymal stem cells

Abstract

The present invention aims to provide a method for producing a cell preparation for joint therapy, a method for culturing mesenchymal stem cells, and a cell preparation for joint therapy produced by the above method, which can improve the cell proliferation rate, reduce the required serum volume, and suppress karyotype abnormalities during the culture process. [Solution] The present invention provides a method for producing a cell preparation for joint treatment, comprising culturing mesenchymal stem cells in a culture medium containing ascorbic acid-2-phosphate trisodium, alanylglutamine, and pyridoxine.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a cell preparation for articular treatment, which comprises culturing mesenchymal stem cells in a medium containing predetermined components, and to a method for culturing mesenchymal stem cells. The present invention further relates to a cell preparation for articular treatment produced by the above method. [Background Art]

[0002] In the field of orthopedic surgery, articular cartilage damage and meniscal injury are frequently encountered in daily clinical practice and are widely recognized as conditions affecting a large number of patients. When articular cartilage damage or meniscal injury occurs, it causes symptoms such as joint pain, reduced range of motion, articular hydrops, and movement disorders. Patients with articular cartilage damage or meniscal injury caused by trauma usually receive treatment from an orthopedic surgeon. Surgical treatment for cartilage damage or meniscal injury aims to remove debris that causes further deterioration of the joint and restore the function of the affected joint. However, it is generally known that cartilage and meniscal tissues have difficulty in self-regeneration.

[0003] On the other hand, with the advancement of regenerative medicine technology in recent years, cell therapy capable of repairing cartilage and meniscus has been actively developed. Among these, mesenchymal stem cells (MSCs) are expected to be a useful cell source for cell therapy. It has been reported that mesenchymal stem cells can be collected from various body tissues and isolated from bone marrow tissue, adipose tissue, muscle tissue, synovial tissue, periosteal tissue, and the like. (Non-Patent Document 1) In particular, it has been reported that synovium-derived mesenchymal stem cells have higher proliferation ability and chondrogenic ability compared to mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow (Non-Patent Document 2). In addition, Patent Document 1 and Patent Document 2 disclose methods for treating articular cartilage damage and meniscal injury using synovium-derived mesenchymal stem cells. [Prior Art Documents] [Non-Patent Documents]

[0004] [Non-Patent Document 1] Na Li, et al., 2020, Stem Cell Research & Therapy. 11:381 [Non-Patent Document 2] Sakaguchi, et al., 2005, Arthritis Rheum. 52:2521-9 [Patent Document]

[0005] [Patent Document 1] Japanese Patent No. 5928961 Publication [Patent Document 2] Japanese Patent No. 5656183 Publication [Summary of the Invention] [Problem to be Solved by the Invention]

[0006] It has become clear that karyotypic abnormalities may occur during cell culture in stem cells including mesenchymal stem cells. In cell transplantation for articular treatment, it is necessary to reduce karyotypic abnormalities to ensure safety. In addition to ensuring safety, it is also necessary to secure a sufficient number of cells to achieve therapeutic effects. Furthermore, when autologous serum is used in autologous therapy, it is also necessary to reduce the amount of serum since the amount of collectable serum is limited.

[0007] An object of the present invention is to solve the problem of providing a method for producing a cell preparation for articular treatment, a method for culturing mesenchymal stem cells, and a cell preparation for articular treatment produced by said method, which can improve the cell proliferation fold, reduce the required amount of serum, and suppress karyotypic abnormalities during the culture process. [Means for Solving the Problem]

[0008] The inventors of this invention diligently studied to solve the above problems and found that the above problems can be solved by culturing mesenchymal stem cells in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine. This invention was completed based on the above findings.

[0009] In other words, the present invention provides the following invention. <1> A method for producing a cell preparation for joint treatment, comprising culturing mesenchymal stem cells in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine. <2> The above mesenchymal stem cells are synovial-derived mesenchymal stem cells. <1> Methods used. <3> The above mesenchymal stem cells are of autologous origin. <1> or <2> Methods used. <4> The above culture medium contains the same type of serum. <1> from <3> The method described in any one of the following ways. <5> The above culture medium does not contain ascorbic acid. <1> from <4> The method described in any one of the following ways. <6> The above culture medium contains one or more of either biotin or lipoic acid. <1> from <5> The method described in any one of the following ways. <7> Mesenchymal stem cells are cultured using multilayer flasks with five or more layers. <1> from <6> The method described in any one of the following ways. <8> The method further includes separating a tissue suspension containing mesenchymal stem cells into two layers, an upper layer and a lower layer, and collecting the lower layer of the two layers, The mesenchymal stem cells contained in the lower layer are cultured in a medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine. <1> from <7> The method described in any one of the following ways. <9> <1> from <8> A cell preparation for joint treatment manufactured by any one of the methods described above. <10> Free from extracellular matrix or scaffold, <9> Cell preparations for joint treatment as described above. <11> It is a meniscus treatment or osteoarthritis treatment. <9> or <10> Cell preparations for joint treatment as described above. <12> Mesenchymal stem cells are human mesenchymal stem cells, and they have 46 chromosomes, with chromosome karyotyping consisting of a pair of chromosomes 1 through 22 and either XX or XY chromosomes. <9> from <11> A cell preparation for joint treatment described in any one of the following. <13> The process of separating a suspension of tissue containing mesenchymal stem cells into two layers, an upper layer and a lower layer. To collect the lower layer of the two layers mentioned above, and A method for culturing mesenchymal stem cells, comprising culturing the mesenchymal stem cells contained in the lower layer in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine. <14> The above mesenchymal stem cells are synovial-derived mesenchymal stem cells. <13> Methods used. <15> The above mesenchymal stem cells are of autologous origin. <13> or <14> Methods used. <16> The above culture medium contains the same type of serum. <13> from <15> The method described in any one of the following ways. <17> The above culture medium does not contain ascorbic acid. <13> from <16> The method described in any one of the following ways. <18> The above culture medium contains one or more of either biotin or lipoic acid. <13> from <17> The method described in any one of the following ways. <19> Mesenchymal stem cells are cultured using multilayer flasks with five or more layers. <13> from <18> The method described in any one of the following ways. <20> <13> from <19> A cell preparation for joint treatment comprising mesenchymal stem cells cultured by any one of the methods described herein. <21> Free from extracellular matrix or scaffold, <20> Cell preparations for joint treatment as described above. <22> It is a meniscus treatment or osteoarthritis treatment. <20> or <21> Cell preparations for joint treatment as described above. <23> Mesenchymal stem cells are human mesenchymal stem cells, and they have 46 chromosomes, with chromosome karyotyping consisting of a pair of chromosomes 1 through 22 and either XX or XY chromosomes. <20> from <22> A cell preparation for joint treatment described in any one of the following.

[0010] A step of transplanting a cell preparation for joint treatment produced by the method of the present invention or mesenchymal stem cells cultured by the method of the present invention so as to cover the cartilage injury site or meniscus injury site with mesenchymal stem cells; and A process of regenerating cartilage tissue in situ at the site of cartilage damage or meniscus damage by differentiating mesenchymal stem cells into chondrocytes; Treatment methods for joints, including [Effects of the Invention]

[0011] According to the present invention, when producing a sufficient amount of cells from tissue, the proliferation rate can be improved, the required amount of serum can be reduced, and karyotype abnormalities can be suppressed during the culture process. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the proliferation curves of mesenchymal stem cells when using type medium or medium A. [Figure 2] Figure 2 shows the karyotype analysis of mesenchymal stem cells using type medium or medium A. [Figure 3] Figure 2 shows the karyotype analysis of mesenchymal stem cells using a medium supplemented with bFGF as the type medium, or using medium A. [Figure 4] Figure 4 shows images of the medial menisci of both knee joints extracted from rats administered with rat synovial mesenchymal stem cells (rSMSCs) or PBS. [Modes for carrying out the invention]

[0013] The details of the present invention will be described below. In this specification, "~" is used to mean that the numbers before and after it are included as the lower limit and upper limit.

[0014] <1> Method for manufacturing cell-based joint therapy products The present invention relates to a method for producing a cell preparation for joint treatment, comprising culturing mesenchymal stem cells in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine. By culturing mesenchymal stem cells in a culture medium containing the above-mentioned specific components, high cell proliferation can be obtained despite the suppression of the occurrence of karyotype abnormalities. In other words, the ability to achieve both suppression of karyotype abnormalities and high cell proliferation is not something that could be predicted from conventional knowledge and is a completely unexpected result.

[0015] The method for producing a cell preparation for joint therapy according to the present invention may include culturing mesenchymal stem cells in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine, and producing a cell preparation for joint therapy using the mesenchymal stem cells cultured as described above.

[0016] <Mesenchymal stem cells> Mesenchymal stem cells broadly refer to a population of stem cells or their progenitor cells that are capable of differentiating into all or some of the mesenchymal cells, such as osteoblasts, chondrocytes, lipoblasts, and muscle cells. Mesenchymal stem cells are known to be present in bone marrow, synovial membrane, periosteum, adipose tissue, and muscle tissue. In relation to the differentiation of mesenchymal stem cells into chondrocytes, it is known that adding BMP (bone morphogenetic protein) or TGF-β (Transforming growth factor-β) to the culture medium promotes the differentiation of undifferentiated mesenchymal stem cells into chondrocytes, and that cartilage tissue can be regenerated under in vitro conditions.

[0017] Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and small molecule compounds. Examples of molecules characteristic of mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers) such as CD73, CD90, CD105, and CD166. Examples of negative markers not expressed in mesenchymal stem cells include, but are not limited to, CD19, CD34, CD45, HLA-DR, CD11b, and CD14. CD stands for Clusters of differentiation, and HLA-DR stands for human leukocyte antigen-D-related. These positive and negative markers can be used to confirm the presence of mesenchymal stem cells. Immunological methods can be used to detect these markers, but detection may also be performed by quantifying the mRNA levels of each molecule.

[0018] The animal species from which mesenchymal stem cells are derived is not particularly limited; for example, they may be cells from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cats, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The mesenchymal stem cells are preferably human mesenchymal stem cells.

[0019] The origin of the mesenchymal stem cells is not particularly limited, but is preferably derived from synovial membrane, bone marrow, fat, dental pulp, fetal cells, or induced pluripotent stem cells. More preferably, the mesenchymal stem cells are synovial membrane-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells, and even more preferably synovial membrane-derived mesenchymal stem cells.

[0020] The mesenchymal stem cells may be autologous or allogeneic, but autologous is preferred. Mesenchymal stem cells may be genetically modified cells or non-genetically modified cells, but non-genetically modified cells are preferred.

[0021] Mesenchymal stem cells can be collected from the above-mentioned tissues by conventional methods. Preferably, mesenchymal stem cells can be collected by separating the suspension of tissue containing mesenchymal stem cells into two layers, an upper and a lower layer, and collecting the lower layer. Separating the suspension of tissue containing mesenchymal stem cells into two layers, an upper and a lower layer, can be done, for example, by centrifugation.

[0022] As an example, we will explain a method for collecting synovial-derived mesenchymal stem cells from synovial tissue. Synovial tissue can be collected from the non-weight-bearing portion of the joint under anesthesia. The amount of synovial tissue collected can be determined considering the type of donor or the amount of synovial-derived mesenchymal stem cells required. For example, synovial-derived mesenchymal stem cells can be obtained from 0.1g to 10g, preferably 0.1g to 2.0g, more preferably 0.1g to 1.5g, and even more preferably 0.1g to 1.0g of synovial tissue. The collected synovial tissue is shredded with scissors or the like as needed and then subjected to enzymatic treatment. The enzyme is not particularly limited as long as it contains a protease, but preferably it is a mixed enzyme containing one or more collagenases and one or more neutral proteases. A particularly preferred enzyme is liberase. As liberase, for example, liberase MNP-S (manufactured by Roche) can be used, which is an enzyme containing collagenase class I, collagenase class II, and a neutral protease (thermocillin). The enzyme concentration in the enzyme treatment is preferably 0.01 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 10 mg / ml, even more preferably 0.5 mg / ml to 10 mg / ml, even more preferably 0.5 mg / ml to 5.0 mg / ml, particularly preferably 0.5 mg / ml to 2.0 mg / ml, and most preferably 0.7 mg / ml to 2.0 mg / ml. The mass ratio of synovial tissue to enzyme is preferably 1000:1 to 10:1, more preferably 500:1 to 20:1, and even more preferably 200:1 to 40:1.

[0023] The enzymatic reaction can be carried out at a temperature of preferably 15°C to 40°C, more preferably 20°C to 40°C. The reaction time should be 30 minutes or more, preferably 2 hours or more, more preferably 2 hours and 30 minutes or more, and even more preferably 3 hours or more. There is no particular upper limit to the reaction time, but it is generally within 4 hours. The enzymatically treated mixture contains synovial mesenchymal stem cells. The enzymatically treated mixture can be transferred to a centrifuge tube through a cell strainer, and the synovial mesenchymal stem cells can be recovered by centrifugation.

[0024] <culture medium> The culture medium used in the present invention preferably contains essential amino acids. Specifically, the culture medium preferably contains histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The concentration of essential amino acids in the culture medium is not particularly limited, but the concentration of each essential amino acid is preferably 0.003 mmol / L or higher, more preferably 0.005 mmol / L or higher, and even more preferably 0.01 mmol / L or higher. The upper limit is generally 5 mmol / L or lower. The total concentration of essential amino acids is preferably 0.5 mmol / L or higher, more preferably 1 mmol / L or higher, and even more preferably 1.5 mmol / L or higher. The upper limit is generally 15 mmol / L or lower.

[0025] The culture medium preferably contains non-essential amino acids, etc. Here, "non-essential amino acids, etc." refers to non-essential amino acids and glutamines. Examples of non-essential amino acids, etc. include one or more selected from the group consisting of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamines, glutamic acid, proline, serine, and tyrosine.

[0026] When non-essential amino acids such as glycine, alanine, serine, proline, asparagine, aspartic acid, glutamines, and / or glutamic acid are included, each of these is present in a concentration of 0.005 mmol / L (5 μmol / L) or higher, preferably 0.01 mmol / L or higher, and more preferably 0.05 mmol / L or higher. The upper limit is generally 3 mmol / L or lower. Alanylglutamine is a preferred example of glutamines. However, the culture medium used in this invention contains alanylglutamine as an essential component.

[0027] The total concentration of non-essential amino acids other than alanylglutamine is preferably 0.5 mmol / L or higher, more preferably 1.5 mmol / L or higher, and even more preferably 2.5 mmol / L or higher. The upper limit is generally 30 mmol / L or lower.

[0028] The concentration of alanylglutamine is preferably 0.5 mmol / L or higher, more preferably 1.0 mmol / L or higher, and even more preferably 1.5 mmol / L or higher. The upper limit is generally 10 mmol / L or lower.

[0029] The culture medium preferably does not contain glutamine. Here, "not containing" means that it is substantially absent in relation to the effects of the present invention, and does not mean that unavoidable contamination is excluded. An example of a substantially absent culture medium is one that typically contains 5 × 10 -5 The concentration is less than mmol / L, preferably 0 mmol / L.

[0030] The detection and measurement methods for amino acids may be those known, such as quantitative analysis of amino acids by high-performance liquid chromatography (HPLC) or amino acid analysis by the ninhydrin method (see, for example, Clinical Chemistry (1997), Vol. 43, No. 8, pp. 1421-1428).

[0031] The amino acids described herein may be L-, D-, or DL-forms. Furthermore, amino acids may exist not only as free forms but also as salts. Examples of salt forms include acid addition salts and salts with bases. Examples of acids include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid, lactic acid, citric acid, tartaric acid, maleic acid, fumaric acid, or monomethylsulfuric acid. Examples of bases that form such salts include metal hydroxides or carbon oxides such as sodium, potassium, and calcium, inorganic bases such as ammonia, and organic bases such as ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. The salts may also be hydrates (hydrated salts).

[0032] The culture medium contains pyridoxine. The culture medium may contain biotin. The culture medium may further contain at least one other vitamin in addition to pyridoxine and biotin. Other vitamins include vitamin B12, choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxals (excluding pyridoxine), riboflavin, thiamine hydrochloride, and i-inositol. Pyridoxal is an example of a pyridoxal. The pyridoxine, biotin, and at least one other vitamin mentioned above may be in free form or form salts. Examples of salt forms include acid addition salts and salts with bases. Specifically, the amino acids mentioned above are examples.

[0033] The concentrations of pyridoxine, biotin, and at least one other vitamin in the culture medium are preferably 0.00005 mmol / L or higher, more preferably 0.0001 mmol / L or higher, and even more preferably 0.0002 mmol / L or higher. The upper limit is generally 1 mmol / L or lower. The total concentration of vitamins in the culture medium is preferably 0.001 mmol / L or more, more preferably 0.005 mmol / L or more, and even more preferably 0.01 mmol / L or more. The upper limit is generally 2 mmol / L or less.

[0034] The culture medium preferably does not contain pyridoxal or pyridoxal derivatives (excluding pyridoxine). Here, "not contained" means substantially absent in relation to the effects of the present invention, and does not mean excluding unavoidable contamination. An example of substantially absent pyridoxal is typically 5 × 10⁻⁶. -5 The concentration is less than mmol / L, preferably 0 mmol / L.

[0035] The culture medium preferably contains at least one inorganic salt. The inorganic salt is preferably one or more selected from the group consisting of calcium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate. The concentration of the inorganic salt is not particularly limited, but the total is preferably 10 mmol / L or more, more preferably 50 mmol / L or more, and even more preferably 80 mmol / L or more. The upper limit is generally 1,000 mmol / L or less.

[0036] The culture medium preferably contains at least one of sugars and pyruvate. Examples of sugars include D-glucose. Examples of pyruvate include sodium pyruvate. The total concentration of sugars and pyruvate is preferably 0.1 mmol / L or more, more preferably 0.3 mmol / L or more, and even more preferably 1 mmol / L or more. The upper limit is generally 50 mmol / L or less.

[0037] The culture medium preferably contains lipoic acid. The concentration of lipoic acid is 5 × 10⁻⁶. -5It is preferable that the concentration be mmol / L or higher, more preferably 0.0001 mmol / L or higher, and even more preferably 0.0005 mmol / L or higher. The upper limit is generally 0.005 mmol / L or lower.

[0038] The culture medium used in this invention contains an ascorbic acid derivative instead of ascorbic acid. Examples of ascorbic acid derivatives include ascorbic acid-2-phosphate, trisodium ascorbic acid-2-phosphate ester, magnesium ascorbic acid-2-phosphate ester, and ascorbic acid-2-glycoside. One of these may be selected and used, or two or more may be used in combination. It is preferable to use trisodium ascorbic acid-2-phosphate ester. The culture medium used in this invention preferably does not contain ascorbic acid. [ka]

[0039] In this invention, it is important to include an ascorbic acid derivative instead of ascorbic acid. This is because the stability of the culture medium is improved by using an ascorbic acid derivative instead of the unstable ascorbic acid.

[0040] In the culture medium composition of the present invention, the total concentration of the ascorbic acid derivative is preferably 0.03 mmol / L or more, more preferably 0.1 mmol / L or more, and even more preferably 0.14 mmol / L or more. The upper limit is preferably 5.0 mmol / L or less, more preferably 1.0 mmol / L or less, and even more preferably 0.57 mmol / L or less.

[0041] The culture medium preferably does not contain linoleic acid. Furthermore, the culture medium preferably does not contain nucleic acids. Here, "not included" means that it is substantially not included in relation to the effects of the present invention, and does not mean that substances that are inevitably included are excluded. An example of a substance that is substantially not included is ascorbic acid, which is typically 5 × 10 -5 The concentration is less than mmol / L, preferably 0 mmol / L. In the case of linoleic acid, it is usually less than 0.0015 mmol / L, preferably 0.001 mmol / L or less, more preferably 0.0005 mmol / L or less, even more preferably 0.0003 mmol / L or less, particularly preferably 0.00015 mmol / L or less, and most preferably 0 mmol / L. In the case of nucleic acids, it is usually 5 × 10 -5 The concentration is less than mmol / L, preferably 0 mmol / L.

[0042] It is known that mesenchymal stem cells can be differentiated into chondrocytes and used to produce cartilage tissue in vitro by culturing them in a cartilage-forming medium supplemented with transforming growth factor β3 (TGF-β3), dexamethasone, and bone morphogenetic factor 2 (BMP-2). Therefore, in order to prevent mesenchymal stem cells from differentiating into chondrocytes, it is preferable that the culture medium does not contain TGF-β3, dexamethasone, and BMP-2.

[0043] Here, "not included" means substantially absent, and does not mean excluding substances that are inevitably present. Examples of substances substantially absent include TGF-β3, which is usually less than 0.1 ng / mL, preferably 0 ng / mL; dexamethasone, which is usually less than 1.0 nmol / L, preferably 0 nmol / L; and BMP-2, which is usually less than 0.1 ng / mL, preferably 0 ng / mL.

[0044] The culture medium preferably does not contain insulin, transferrin, selenite, or BSA (bovine serum albumin). Here, "not included" means not included in practice, and does not mean excluding substances that are inevitably present. An example of something not included in practice is insulin, which is usually... The concentration is less than 10 ng / mL, preferably 0 ng / mL. In the case of transferrin, it is usually less than 10 ng / mL, preferably 0 ng / mL. In the case of selenite, it is usually less than 0.01 ng / mL, preferably 0 ng / mL. In the case of BSA, it is usually less than 10 μg / mL, preferably 0 μg / mL.

[0045] The culture medium may contain phenol red. The concentration of phenol red is preferably 0.001 mmol / L or higher, more preferably 0.005 mmol / L or higher, and even more preferably 0.01 mmol / L or higher. The upper limit is generally 0.2 mmol / L or lower.

[0046] The culture medium may be one containing serum or one without serum. In a serum-containing medium, the lower limit of serum content is preferably 2% by volume or more, more preferably 5% by volume or more, and even more preferably 10% by volume or more. The upper limit is generally 20% by volume or less.

[0047] Examples of serum include animal-derived serum, but human serum is preferred.

[0048] The serum can be either allogeneic or heterogeneic, but allogeneic serum is preferred. That is, when producing mesenchymal stem cells from human tissue for the purpose of administration to humans, a culture medium containing human serum may be used. When using allogeneic serum, it can be autologous serum or allogeneic, heterogeneous serum, but autologous serum is preferred.

[0049] The culture medium may further contain antibiotics. Examples of antibiotics include streptomycin, gentamicin (such as gentamicin sulfate), penicillin, and amphotericin B. The total concentration of the antibiotics is preferably 0.1 mg / L or higher, more preferably 0.5 mg / L or higher, and even more preferably 10.0 mg / L or higher. The upper limit is generally 1000 mg / L or lower.

[0050] In addition to the components mentioned above, the culture medium may contain known additives as needed. Examples of additives include polyamines (e.g., putrescine), reducing agents (e.g., 2-mercaptoethanol), and buffering agents (e.g., HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)).

[0051] <Culture method> The culture vessel used for culturing mesenchymal stem cells is not particularly limited as long as it is capable of culturing mesenchymal stem cells, but examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles. Preferably in the present invention, mesenchymal stem cells may be cultured using multi-layer flasks with 5 or more layers (for example, 5 to 10 layers). An example of a multi-layer flask with 5 or more layers is a 10-cell stack (polystyrene cell stack-10 chamber, manufactured by Corning). Flasks equipped with vent caps can also be used. By using a flask equipped with a vent cap and culturing while forcibly passing gas through the vent cap, the proliferation rate of mesenchymal stem cells can be significantly improved.

[0052] The culture vessel may be cell-adherent or non-cell-adherent, and the appropriate choice depends on the purpose. Cell-adherent culture vessels may be coated with any cell-supporting substrate, such as extracellular matrix (ECM), to improve the adhesion of the culture vessel surface to cells. The cell-supporting substrate may be any substance intended for the adhesion of mesenchymal stem cells, such as Matrigel using ECM, or collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, or fibronectin.

[0053] The culture conditions can be set as appropriate. For example, the culture temperature is not particularly limited but may be about 30-40°C, preferably about 37°C. The CO2 concentration may be about 1-10%, preferably about 2-5%. The oxygen concentration may be 1-20%, preferably 1-10%.

[0054] It is preferable to adjust the culture period in order to proliferate mesenchymal stem cells in an undifferentiated state and with good in situ chondrogenesis ability. It is also necessary to consider the need to prepare a sufficient number of undifferentiated mesenchymal stem cells to cover the cartilage damage and regenerate the affected area. Therefore, the culture period for mesenchymal stem cells is preferably 5 days or more, 7 days or more, or 10 days or more, and may be 10-100 days, 10-90 days, 10-80 days, 10-70 days, 10-60 days, 10-50 days, 10-40 days, 10-30 days, 10-28 days, 10-21 days, or 10-14 days. When using synovial-derived mesenchymal stem cells as mesenchymal stem cells, it is preferable to adjust the culture period in order to proliferate synovial-derived mesenchymal stem cells in an undifferentiated state and with good in situ chondrogenesis ability. It is also necessary to consider the need to prepare a sufficient number of undifferentiated synovial-derived mesenchymal stem cells to cover the cartilage damage and regenerate the affected area. Therefore, the culture period for synovial-derived mesenchymal stem cells is preferably 5 days or more, 7 days or more, or 10 days or more, and may be 10 to 100 days, 10 to 90 days, 10 to 80 days, 10 to 70 days, 10 to 60 days, 10 to 50 days, 10 to 40 days, 10 to 30 days, 10 to 28 days, 10 to 21 days, or 10 to 14 days.

[0055] It is known that the in situ chondrogenic ability of mesenchymal stem cells decreases inversely proportional to the number of passages in the mesenchymal stem cells in vitro. Therefore, in order to prepare undifferentiated mesenchymal stem cells, it is preferable to have 10 passages or less, more preferably 5 passages or less, and even more preferable to produce primary or first-passage mesenchymal stem cells. It is known that synovial-derived mesenchymal stem cells (MSCs) exhibit a decrease in in situ chondrogenesis in inverse proportion to the number of passages in vitro. Therefore, to prepare undifferentiated MSCs, it is preferable to have 10 passages or less, and more preferably 5 passages or less.

[0056] <2> Methods for culturing mesenchymal stem cells The present invention The process of separating a suspension of tissue containing mesenchymal stem cells into two layers, an upper layer and a lower layer. To collect the lower layer of the two layers mentioned above, and To culture the mesenchymal stem cells contained in the lower layer in a medium containing ascorbic acid-2-phosphate trisodium, alanylglutamine, and pyridoxine, This relates to a method for culturing mesenchymal stem cells, including [specific example]. Specific examples and preferred embodiments of the mesenchymal stem cells, culture medium, and culture method in the mesenchymal stem cell culture method according to the present invention described above are as stated herein.

[0057] <3> Cell therapy for joint treatment According to the present invention, a cell preparation for joint treatment manufactured using the cell preparation for joint treatment according to the present invention is provided. Furthermore, according to the present invention, a cell preparation for joint treatment is provided which includes mesenchymal stem cells cultured by the method for culturing mesenchymal stem cells according to the present invention. According to the present invention, a cell preparation for joint treatment containing mesenchymal stem cells obtained by the method of the present invention as an active ingredient can be manufactured.

[0058] Cell-based therapies for joint treatment preferably do not contain extracellular matrix or scaffolds. Examples of extracellular matrix or scaffolds used in this context include collagen, hyaluronic acid, alginic acid, polylactic acid, and polyglycolic acid.

[0059] For mesenchymal stem cells used as the active ingredient in cell therapies for joint treatment, it is preferable that the cells are free from karyotype abnormalities. Examples of cells free from karyotype abnormalities include human mesenchymal stem cells, which have 46 chromosomes and whose chromosome karyotyping consists of a pair of chromosomes 1 through 22 and either XX or XY chromosomes.

[0060] The mesenchymal stem cells used as the active ingredient in cell preparations for joint therapy preferably have a proportion of normal cells that do not contain the karyotype abnormalities described above, which is more preferably higher than 90%, more preferably 91% or higher, even more preferably 93% or higher, even more preferably 95% or higher, even more preferably 98% or higher, particularly preferably 99% or higher, and most preferably 100%.

[0061] Joint treatments include the treatment of diseases involving joint injury, damage, or inflammation, and can include joint diseases resulting from degeneration and / or inflammation of connective tissue such as cartilage, or non-inflammatory joint diseases. Joint treatments can include, but are not limited to, the treatment of diseases selected from the group consisting of meniscal tears, traumatic cartilage injuries, osteochondritis dissecans, avascular osteonecrosis, osteoarthritis, rheumatoid arthritis (e.g., chronic rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defects. Osteoarthritis may be knee osteoarthritis, or it may be elbow, finger, hip, shoulder, ankle, or cervical spine, or a combination of multiple joint sites. The cell preparation for joint treatment of the present invention is preferably a meniscal treatment agent or an osteoarthritis treatment agent.

[0062] When manufacturing cell preparations for joint therapy, a conventional method may be used to prepare a preparation suitable for administration to an individual, such as by mixing mesenchymal stem cells with a pharmaceutically acceptable carrier. Examples of carriers include saline solution, glucose, and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) to make it isotonic for injection. Furthermore, buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. may also be added.

[0063] [Treatment methods for joints] The present invention further relates to a method for treating a joint. More specifically, the present invention relates to a method for treating a disease selected from the group consisting of meniscus injury, traumatic cartilage injury, osteochondritis dissecans, avascular necrosis, osteoarthritis (e.g., knee osteoarthritis where the joint site is the knee, wherein the joint site is selected from elbow joint, finger joint, hip joint, shoulder joint, ankle joint, cervical vertebra, and a combination of a plurality of joint sites), rheumatoid arthritis (e.g., chronic rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defect.

[0064] The method for treating a joint of the present invention comprises: a step of implanting the joint therapeutic agent of the present invention such that a cartilage injury site or a meniscus injury site is covered with mesenchymal stem cells; and a step of regenerating cartilage tissue in situ at the cartilage injury site or the meniscus injury site by differentiating the mesenchymal stem cells into chondrocytes; .

[0065] When implanting the joint therapeutic agent of the present invention into a patient, in order to efficiently treat a cartilage injury site or a meniscus injury site, 1×10 6 to 1.0×10 11 cells, 2×10 6 to 1.0×10 11 cells, 5×10 6 to 1.0×10 11 cells, 1×10 7 to 1.0×10 11 cells, 2.0×10 7 to 1.0×10 11 cells, 2.5×10 7 to 1.0×10 11 cells, 3.0×10 7 to 1.0×10 11 cells, 4.0×10 7 to 1.0×10 11 cells, 1×10 6 to 1.0×10 10 cells, 2.5×10 7 to 1.0×10 10 cells, 1×10 6 to 1.0×10 9 cells, 2.5×10 7 to 1.0×10 9pieces, 1×10 6 ~1.0×10 8 pieces, 2.5×10 7 ~1.0×10 8 1, or 2.0 × 10 7 ~1.0×10 8 Individual mesenchymal stem cells, more preferably 2.0 × 10⁶ 7 ~1.0×10 8 It is preferable to apply individual mesenchymal stem cells.

[0066] By transplanting mesenchymal stem cells into the cartilage or meniscus injury site, the site becomes covered with mesenchymal stem cells. Mesenchymal stem cell transplantation can be performed by open surgery or arthroscopic surgery. To minimize invasiveness, arthroscopic transplantation of mesenchymal stem cells is preferred.

[0067] The cartilage or meniscus injury may be covered with a suspension of mesenchymal stem cells or with a cell sheet of mesenchymal stem cells. Mesenchymal stem cells have a high ability to adhere to cartilage or meniscus injuries.

[0068] In the treatment of cartilage damage, the minimally invasive procedure of the present invention is characterized by covering the cartilage damage site with mesenchymal stem cells, and involves the following steps: Maintain a position that faces upwards towards the cartilage injury; Placing a cell sheet of mesenchymal stem cells, a suspension of mesenchymal stem cells, or a gel-like substance containing mesenchymal stem cells on the surface of the cartilage injury; and Maintaining a specific position for a certain period of time to allow mesenchymal stem cells to adhere to the surface of the cartilage injury; Includes.

[0069] In the treatment of meniscus injuries, the minimally invasive procedure of the present invention is characterized by covering the meniscus injury site with mesenchymal stem cells, and involves the following steps: Maintain a position where the injured meniscus is facing downwards; Injecting a suspension of mesenchymal stem cells into the knee joint; and Maintaining a specific body position for a certain period of time to allow mesenchymal stem cells to adhere to the meniscus injury site; Includes.

[0070] To ensure that mesenchymal stem cells adhere securely to the surface of the cartilage or meniscus injury, it is preferable to hold the transplanted mesenchymal stem cells on the surface of the cartilage or meniscus injury for at least 10 minutes, preferably 15 minutes. To achieve this, the body position is maintained for at least 10 minutes, preferably 15 minutes, with the cartilage or meniscus injury facing upwards and the mesenchymal stem cells being held on the upward-facing cartilage or meniscus injury.

[0071] The cartilage or meniscus injury site, which contains mesenchymal stem cells, can be further covered with periosteum to strengthen the adhesion of the mesenchymal stem cells to the cartilage or meniscus injury site. The surgery is completed after the mesenchymal stem cells are retained on the surface of the cartilage or meniscus injury site for at least 10 minutes.

[0072] In this invention, transplanted mesenchymal stem cells differentiate into chondrocytes at the site of cartilage damage or meniscal damage, and then regenerate cartilage tissue in situ at the site of cartilage damage or meniscal damage.

[0073] During the in situ chondrogenesis process of mesenchymal stem cells, cartilage tissue regenerates according to the local microenvironment (nutrient supply and cytokine environment, etc.), so no external intervention is required. As a result of in situ chondrogenesis by synovial-derived mesenchymal stem cells, cartilage tissue is regenerated at the site of cartilage damage or meniscal damage, repairing the damage. In the case of cartilage damage, the bone region, the boundary between cartilage and bone, the cartilage center, the surface region, and the region adjacent to the original cartilage are formed as the original cartilage tissue, or in the case of meniscal damage, meniscal cartilage is formed.

[0074] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0075] [Experiment A] <Materials and Methods> (1)Cell Mesenchymal stem cells (MSCs) isolated from human bone marrow tissue (Whole Bone Marrow, Fresh, 10 mL, Model No.: ALL-ABM001, Lot: B009, B012) purchased from Allcells were used.

[0076] (2) Basal culture medium Type of basal medium: Thermo Fisher Scientific MEM alpha no nucleosides (Model number: 12561)

[0077] Basic culture medium A: The type medium was modified by replacing ascorbic acid, glutamine, and pyridoxal with trisodium ascorbic acid-2-phosphate, alanylglutamine, and pyridoxine, respectively.

[0078] Table 1 shows the media composition of Type basal medium and basal medium A.

[0079] [Table 1] JPEG2026140988000003.jpg252170JPEG2026140988000004.jpg35170

[0080] (3) Culture of mesenchymal stem cells For culturing mesenchymal stem cells, 20 μg / mL of gentamicin sulfate ("Takada Pharmaceutical Gentamicin Injection 60") and fetal bovine serum (SAFC, model no. 12007C or Selforn, model no. FBS-04) were added to each of the basal media described in (2) to a final concentration of 15% (v / v). Hereinafter, these will be referred to as type medium and medium A, respectively. In addition, to improve proliferation, bFGF (Fiblast Spray 250 (Kaken Pharmaceutical) dissolved in sterile water for injection and prepared to 10 μg / mL) was added to the above type medium to a final concentration of 10 ng / mL. Hereinafter, this will be referred to as type medium + bFGF. bFGF is an abbreviation for basic fibroblast growth factor.

[0081] To isolate mesenchymal stem cells, centrifuge the tissue suspension of purchased human bone marrow tissue (lots: B009, B012) at 1,000 rpm for 10 minutes to separate it into two layers. Collect and use the lower layer. Add type medium, medium A, or type medium + bFGF to this lower layer, after removing the plasma, to 10 times the volume before plasma removal, at a concentration of 0.20 mL / cm³. 2 The cells were seeded in a flask and cultured in an incubator at 37°C under a 5% CO2 atmosphere. The culture medium was changed every 3 or 4 days, and after 12 to 13 days of culture from seeding, the cells were detached with a 0.05% trypsin-EDTA solution (hereinafter, trypsin, Life Technologies, model number: 25300). After detachment, the cells were neutralized with an equal volume of culture medium and transferred to a separately prepared tube. The culture flask was washed again with an equal volume of culture medium and added to the above tube, and the remaining cells were collected. The tube was centrifuged at 200 × g for 5 minutes, and the supernatant was removed. An appropriate amount of culture medium was added to the remaining pellet, and the cell count was performed using a hemocytometer. The final concentration was 0.20 to 0.50 × 10⁶. 4 cells / cm 2Seeds were seeded in flasks and cultured in an incubator at 37°C under a 5% CO2 atmosphere. After culturing for 3 or 4 days, when the cells filled more than 60% of the flask bottom, subculturing was repeated using the same procedure as above. For B009, cells were centrifuged at the end of the 9th subculturing, and for B012, cells were centrifuged at the end of the 4th subculturing. The cells were then frozen in a type medium mixed with a freezing solution to obtain a final concentration of 10% (v / v) dimethyl sulfoxide (SIGMA-ALDRICH Model No.: D2650) at a rate of 100 × 10⁶ 4 cells / cm 2 The cells were adjusted to achieve the desired result, and a cell stock was prepared for karyotype analysis and evaluation.

[0082] (4) Karyotype analysis Each prepared cell stock was thawed in a 37°C water bath, centrifuged at 200×g for 5 minutes, the frozen solution was removed, and the respective culture medium was added, resulting in a 0.35~0.50×10 4 cells / cm 2 Seeds were seeded into flasks and cultured for 3 or 4 days in an incubator at 37°C and a 5% CO2 atmosphere. When the cells filled more than 60% of the bottom of the flask, they were detached with trypsin, and the recovered cells were replaced with each culture medium using the method described above, and cultured to 0.25-0.50 × 10⁶. 4 cells / cm 2 The seeds were seeded in a flask and cultured for 3 or 4 days in an incubator at 37°C and a 5% CO2 atmosphere.

[0083] Each culture flask was inoculated with 1 / 100th the volume of demecolsin solution (Fujifilm Wako Pure Chemical Industries, Ltd., model number 045-18761) and left to stand for 3-4 hours in an incubator at 37°C under a 5% CO2 atmosphere. After the reaction was complete, each cell was detached with trypsin. Subsequently, Carnoy fixation was performed according to the method recommended by Trans Chromosomics, and Q-band analysis was requested.

[0084] <Result> (1) Comparison of MSC culture and karyotype analysis using type medium or medium A Frozen stocks of MSCs (P10) that had been passaged 9 times in type medium (lot: B009) and medium A were thawed, and each medium was used for 2 passages. Karyotype analysis was then performed on the P12 cells (passed 11 times). A comparison of proliferation rates revealed that MSCs cultured in medium A exhibited significantly higher proliferation rates compared to those cultured in type medium. Figure 1 shows a comparison of proliferation curves expressed using population doubling values. Population doubling value (PDL) indicates the number of cell divisions and is calculated using the following formula. The PDL was calculated for each passage and accumulated to create the proliferation curve. PDL calculation formula: (Log(number of recovered cells) - Log(number of seeded cells)) / Log2

[0085] Furthermore, karyotype analysis revealed karyotype abnormalities in MSCs cultured in type medium, but no abnormalities were found in MSCs cultured in medium A. The results are shown in Table 2 and Figure 2.

[0086] [Table 2]

[0087] (2) Comparison of MSC culture and karyotype analysis using type medium + bFGF or medium A Frozen stocks (P5) of MSCs (Method Cells) from Lot B012, which had been passaged 4 times in type medium + bFGF and medium A, were thawed, and each medium was used for 2 passages. Karyotype analysis of P7 (passed 6 times) cells was then performed. A comparison of proliferation rates revealed that adding bFGF to the type medium resulted in proliferation comparable to that of medium A. Therefore, karyotype analysis was performed on the type medium + bFGF and medium A. No karyotype abnormalities were found in MSCs cultured in medium A, but karyotype abnormalities were observed in MSCs cultured in type medium + bFGF. These results are shown in Table 3 and Figure 3.

[0088] [Table 3]

[0089] From the results above, it was found that the culture method using medium A can achieve both a very significant improvement in proliferation and normal karyotype on its own.

[0090] [Experiment B] (Materials and Methods) (1)Cell The experiment used either human bone marrow mesenchymal stem cells or porcine synovial mesenchymal stem cells.

[0091] Human bone marrow mesenchymal stem cells were obtained and cultured using the method described in [Experiment A].

[0092] Porcine synovial mesenchymal stem cells were prepared by shredding synovial tissue collected from the knee of a miniature pig (Fuji Micra Co., Ltd.) with scissors and immersing it in 5.0 mL of liberase solution. The liberase solution used was prepared by dissolving 5.0 mg of liberase MNP-S (Roche) in 5.0 mL of sterile water for injection containing 20% ​​final concentration fetal bovine serum (Nichirei Bioscience Co., Ltd., model number: 174012). The enzymatic reaction was carried out at 37°C for 3 hours. Subsequently, the tissue digestate was separated into two layers through a cell strainer, and the lower layer was transferred to a 50 mL centrifuge tube and centrifuged at 400 g for 5 minutes. The supernatant was removed, and the resulting concentrated cell suspension was suspended in culture medium.

[0093] (2) Basal culture medium As an example, the culture medium (Culture Medium A) described in [Experiment A] was used. For comparison, αMEM (Thermo, model number: 12561-056) (type medium) was used.

[0094] (3)Culture container The culture vessel is a T-flask (TPP Corporation cell culture flask, 150 cm²). 2 A filter cap (model number: 90151) or a 10-cell stack (Corning Cell Stack 10 Chamber Cell Culture Surface Treatment, model number: 3270) was used.

[0095] (4) Culture of synovial mesenchymal stem cells For the culture of synovial mesenchymal stem cells, 1% (v / v) of Antibiotic-Antimycotic (Thermo, part number: 15240-062) was added to each basal medium described in (2) as an antibiotic. Autologous serum was added for the culture of human bone marrow mesenchymal stem cells, and fetal bovine serum (Nichirei Bioscience, part number: 174012) was added for the culture of porcine synovial mesenchymal stem cells, with each medium being used to achieve a final concentration of 10-20% (v / v).

[0096] A cell suspension was prepared by suspending cells in the above medium at a predetermined concentration so that the cell seeding density and medium volume after seeding into the culture vessel were in the predetermined conditions, and the predetermined amount was seeded into the culture vessel described in (3). Here, seeding density refers to the number of cells seeded per unit area of ​​the culture surface, and medium volume refers to the volume of medium per unit area of ​​the culture surface. After seeding, the culture vessel was placed in an incubator at 37°C and a 5% CO2 atmosphere and cultured. After culturing for the predetermined period from seeding, the cells were detached from the culture vessel and collected, and the number of cells was measured. The cell proliferation rate was calculated by dividing the number of collected cells after culturing by the number of seeded cells.

[0097] (result) (Experiment 1) Comparison of growth rates based on different culture media Porcine synovial mesenchymal stem cells were cultured in type medium supplemented with 20% (v / v) fetal bovine serum (FBS). Cell seeding densities were 1000 and 1500 cells / cm³. 2 The culture medium volume is 0.12 mL / cm³. 2 T flasks were used as culture vessels. Cells were harvested 12 days after seeding, and their growth rates were calculated. Seeding density: 1000 cells / cm² 2 Table 2 shows the relative growth rates when the growth rate under these conditions is set to 1.

[0098] Under the same conditions as described above for the FBS-supplemented type medium, the culture medium was changed to medium A, and cells were cultured. Cells were collected 12 days after seeding, and the proliferation rate was calculated. Seeding density when using the FBS-supplemented type medium: 1000 cells / cm² 2 Table 4 shows the relative growth rates when the growth rate under these conditions is set to 1.

[0099] In the culture of porcine synovial mesenchymal stem cells, the cell seeding density is 1000-1500 cells / cm³. 2 Within this range, medium A was found to exhibit a higher growth rate compared to type medium.

[0100] [Table 4]

[0101] (Experiment 2) Comparison of growth rates due to differences in culture medium volume 1 Human bone marrow mesenchymal stem cells were cultured using type medium and medium A. In comparative test 1, a medium containing 20% ​​(v / v) FBS was used, with a medium volume of 0.12 mL / cm³. 2 That's what I decided. In comparative test 2, a medium containing 20% ​​(v / v) FBS was used, with a medium volume of 0.24 mL / cm³. 2 That's what I decided. In Test 3, medium A was used with 20% (v / v) FBS added, and the volume of medium was 0.12 mL / cm³. 2 That's what I decided. In Test 4, medium A was used with 20% (v / v) FBS added, and the volume of medium was 0.24 mL / cm³. 2 That's what I decided.

[0102] Cells were collected 12 days after sowing under the conditions of comparative tests 1 and 2, and tests 3 and 4, and the proliferation rate was calculated. Table 5 shows the relative proliferation rates, with the proliferation rate of comparative test 1 set to 1. It was found that increasing the amount of culture medium improved the growth rate in both type medium and medium A.

[0103] [Table 5]

[0104] (Experiment 3) Comparison of growth rates with different amounts of culture medium 2 Porcine synovial mesenchymal stem cells were cultured using type medium and medium A. In comparative test 11, a medium containing 20% ​​(v / v) FBS was used, with a medium volume of 0.12 mL / cm³. 2 That's what I decided. In comparative test 12, a medium containing 20% ​​(v / v) FBS was used, with a medium volume of 0.20 mL / cm³. 2 That's what I decided. In Test 13, medium A was used with 20% (v / v) FBS added, and the volume of medium was 0.12 mL / cm³. 2 That's what I decided. In Test 14, medium A was used with 20% (v / v) FBS added, and the volume of medium was 0.20 mL / cm³. 2 That's what I decided.

[0105] Cells were collected 12 days after sowing under the conditions of comparative tests 11 and 12, and tests 13 and 14, and the proliferation rate was calculated. Table 6 shows the relative proliferation rates, with the proliferation rate of comparative test 1 set to 1. It was found that increasing the amount of culture medium improved the growth rate in both type medium and medium A.

[0106] [Table 6]

[0107] (Test 4) Evaluate proliferation rate without increasing serum usage. Porcine synovial mesenchymal stem cells were cultured using type medium and medium A as the basal medium. In Test 21, a medium containing 20% ​​(v / v) FBS was used, with a medium volume of 0.12 mL / cm³. 2 That's what I decided. In comparative test 22, medium A with 20% (v / v) FBS added was used, and the volume of medium was 0.12 mL / cm³. 2 That's what I decided. In Test 23, to ensure the total amount of FBS used for culture was the same as in Test 21, a medium containing 10% (v / v) FBS was added to medium A, and the volume of medium was 0.24 mL / cm³. 2 That's what I decided.

[0108] Cells were collected 12 days after sowing under the conditions of Test 21, Comparative Test 22, and Test 23, and the proliferation rate was calculated. Table 7 shows the relative proliferation rates, with the proliferation rate of Test 21 set to 1. It is generally known that lowering serum concentration reduces cell proliferation. This is because serum concentration is thought to be the most significant factor in cell proliferation. However, in culture medium A, it was found that even with the same amount of serum used, increasing the amount of basal medium to lower the serum concentration unexpectedly improved the proliferation rate.

[0109] [Table 7]

[0110] (Experiment 5) Effect of serum concentration on proliferation rate Human bone marrow mesenchymal stem cells were cultured using type medium and medium A. In comparative test 31, a type medium supplemented with 20% (v / v) FBS was used, with a medium volume of 0.12 mL / cm³. 2 That's what I decided. In comparative test 32, a type medium with 10% (v / v) FBS added was used so that the total amount of FBS used for culture was the same as in test 31, and the medium volume was 0.24 mL / cm³. 2 That's what I decided. In Test 33, medium A with 10% (v / v) FBS added was used so that the total amount of FBS used for culture was the same as in Test 31, and the volume of medium was 0.24 mL / cm³. 2 That's what I decided.

[0111] The growth rates were evaluated after culturing for 12 days under the conditions of comparative tests 31 and 32, and test 33. The relative growth rates, with the growth rate of test 31 set to 1, are shown in Table 8. In the type medium, even with the same amount of serum used, the growth rate decreases as the serum concentration decreases. However, when medium A is used as the basal medium, it was found that increasing the amount of basal medium and lowering the serum concentration unexpectedly improved the growth rate compared to the type medium. The decrease in growth rate when serum concentration is lowered, as observed in the type medium, is a generally understood phenomenon.

[0112] [Table 8]

[0113] (Experiment 6) Effect of differences in culture vessels on growth rate Porcine synovial mesenchymal stem cells were cultured in T flasks and 10-cell stacks (polystyrene cell stack-10 chamber, Corning). The culture media used were type medium and medium A, each supplemented with 20% (v / v) FBS, at a volume of 0.12 mL / cm³. 2 That's what I decided. In comparative tests 41-1 and 41-2, type medium was used, and cells were cultured using T flasks and 10-cell stacks, respectively. After culturing for 12 days in each culture vessel, the growth rate was evaluated, and the relative growth rate of the 10-cell stacks, with the growth rate of the T flask set to 1, is shown in Table 7. In tests 42-1 and 42-2, type medium was used, and cultures were performed using T flasks and 10-cell stacks, respectively.

[0114] The growth rate was evaluated after culturing in each culture vessel for 12 days, and the relative growth rate of a 10-cell stack, with the growth rate of the T flask set to 1, is shown in Table 9. When the culture vessel was changed from a T-flask to a 10-cell stack, it was found that the growth rate decreased in type medium, while the growth rate improved in medium A.

[0115] [Table 9]

[0116] (Test 7) ​​Effect of culture vessel cap shape and presence or absence of forced ventilation on growth rate Porcine synovial mesenchymal stem cells were cultured in 10-cell stacks. Type medium and medium A, each supplemented with 20% (v / v) FBS, were used, with a medium volume of 0.12 mL / cm³. 2 The following comparison was made for each culture medium: two caps on a 10-cell stack were used, one as a standard filter cap and the other as a vent cap (Corning cell stack transfer cap, model number 3281). In addition, for medium A, the caps were changed to vent caps, and a comparison was made with forced gas ventilation through one of the vent caps. The gas used for forced ventilation was air containing 5% CO2, which was humidified to the same level as the incubator environment (100% relative humidity) and heated to 37°C before being introduced into the 10-cell stack.

[0117] Cells were cultured for 12 days under each culture condition, harvested, and the proliferation rate was calculated. Table 10 shows the relative proliferation rate, with the proliferation result of cells cultured in a T flask using the same amount of medium in type medium set to 1. In the table, cells marked as "culture not continuing" were not cultured for 12 days because it was confirmed that the cells did not proliferate during the culture process.

[0118] When the caps of a 10-cell stack were changed from standard filter caps to vented caps and cultured in type medium, cell growth decreased significantly, making it impossible to continue culturing for the prescribed period. On the other hand, when medium A was used, the decrease in growth rate was suppressed more than with type medium even when the caps were changed from standard filter caps to vented caps, and culturing could be continued. Furthermore, it was found that the growth rate improved significantly when gas was forcibly passed through the caps.

[0119] [Table 10]

[0120] (Experiment 7) Establishment of rat synovial stem cells and meniscus regeneration effect This study demonstrates the meniscus regeneration ability of rat synovial membrane-derived mesenchymal stem cells in rats. Six-week-old Lewis rats were used to establish synovial-derived mesenchymal stem cells. Synovial tissue collected under isoflurane anesthesia was mixed with basal medium A, which contained 20% Fetal Bovine Serum (Gibco Cat.#10270) and 1% Antibiotic-Antimycotic (100X) (Thermofisher Scientific Cat.#15240062), to which 3.0 mg / mL of Collagenase V (Sigma Cat.#C9263) was added. The mixture was reacted at 37°C for 2 hours. The reaction was stopped by adding cooled medium, and the mixture was passed through a 40 μm cell strainer to remove residual tissue. The collected cells were then placed in a 75 cm³ container. 2 Cells were seeded in a cell culture flask (Corning Inc., model number: 353136) and cultured at 37°C for 6 days at a CO2 concentration of 5%. After 6 days of culture, the culture medium in the flask was discarded, and the flask was washed twice with PBS (phosphate-saturated saline). Then, 5 mL of TrypLE® Express (Thermo Fisher Scientific, Cat.#1260413) was added, and the flask was incubated at 37°C for 5 minutes. The cells were collected as mesenchymal stem cells, and the cell count was calculated using a Vi-CELL autoanalyzer (BECKMAN COULTER, model number: 731050). The supernatant was discarded by centrifugation and replaced with COS-banker (Cosmo Bio Co., Ltd., Cat.# COS-CFM01) to create a cell freeze stock.

[0121] To create a meniscus injury model for evaluating the meniscus regeneration effect, 10-week-old Lewis rats were used. The meniscus injury and mesenchymal stem cell transplantation method involved incising the skin of the knee joint under isoflurane anesthesia to expose the knee joint. The medial joint capsule below the patella was exposed, and a longitudinal incision was made with a scalpel to expose the cartilage of the distal femur. The medial meniscus was separated from the synovial membrane, exposing the medial meniscus, and approximately two-thirds of it was resected. The patellar tendon and synovial membrane were sutured together, followed by suture of the muscles. After the meniscus injury procedure, the rats were divided into two groups: a rat synovial-derived mesenchymal stem cell (rSMSC) group and a control group (PBS). In the rSMSC group, the mesenchymal stem cells administered were established and frozen from the synovial membrane, then induced dormancy in basal medium A containing 20% ​​Fetal Bovine Serum (Gibco Cat.#10270) and 1% Antibiotic-Antimycotic (100×) (Thermo Fisher Scientific Cat.# 15240062), cultured for 8 days, and the number of cells harvested was 5×10⁶. 6 Cells were transplanted into the knee joint by inserting a needle perpendicular to the knee joint through the suture site, and the skin was sutured. Postoperatively, all rats were returned to their cages and allowed to exercise and eat freely.

[0122] The animals underwent autopsy three weeks after the procedure. Under isoflurane anesthesia, the animals were euthanized by bleeding through inferior aortic severance. Subsequently, the knee joint was dislocated to expose the meniscus, and the medial meniscus was excised and photographed. Figure 4 shows images of the excised medial menisci of both knee joints. The regenerated portion was identified from the images based on the difference in color and shape from the remaining meniscus and enclosed with a dotted line. In the PBS group, half of the animals had meniscus regeneration up to the middle segment, but in the rSMSC group, many cases showed meniscus regeneration up to the anterior segment, demonstrating the meniscus regeneration effect of rSMSC transplantation.

[0123] To quantitatively compare the meniscus regeneration effect between the rSMSC group and the PBS group, the area of ​​the meniscus regeneration portion (within the dotted line) was measured using ImageJ (version 1.52). The meniscus regeneration portion was visually judged based on the color and shape of the meniscus, and its area was calculated using Equation 1.

[0124] Formula 1: Area of ​​meniscus regeneration (mm 2 ) = Number of pixels in the meniscus regeneration area / 1mm 2 Number of pixels per unit

[0125] The mean, standard deviation, and statistical analysis of the regenerated area for each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analysis was conducted using Student's T-Test between the rSMSC and PBS groups, with a significance level of less than 5% (P<0.05) considered to indicate a difference.

[0126] Table 11 shows the meniscus regeneration area values ​​for the rSMSC group and the PBS group. The meniscus regeneration area for the PBS group was 1.1 mm². 2 In contrast, the rSMSC group showed 2.0 mm 2 It had approximately twice the regeneration area, showing a statistically significant difference (P<0.05).

[0127] [Table 11]

Claims

1. A method for producing a cell preparation for joint treatment, comprising culturing mesenchymal stem cells in a medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine, and lacking extracellular matrix and scaffold.

2. The method according to claim 1, wherein the mesenchymal stem cells are synovial-derived mesenchymal stem cells.

3. The method according to claim 1 or 2, wherein the mesenchymal stem cells are of autologous origin.

4. The method according to any one of claims 1 to 3, wherein the culture medium contains the same type of serum.

5. The method according to any one of claims 1 to 4, wherein the culture medium does not contain ascorbic acid.

6. The method according to any one of claims 1 to 5, wherein the culture medium comprises one or more biotin or lipoic acid.

7. The method according to any one of claims 1 to 6, wherein mesenchymal stem cells are cultured using a multilayer flask with five or more layers.

8. The method further includes separating a tissue suspension containing mesenchymal stem cells into two layers, an upper layer and a lower layer, and collecting the lower layer of the two layers. The method according to any one of claims 1 to 7, wherein mesenchymal stem cells contained in the lower layer are cultured in a culture medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine.

9. A cell preparation for joint treatment, manufactured by the method according to any one of claims 1 to 8.

10. A cell preparation for joint treatment according to claim 9, which is a meniscus treatment agent or an osteoarthritis treatment agent.

11. The cell preparation for joint treatment according to claim 9 or 10, wherein the mesenchymal stem cells are human mesenchymal stem cells, the number of chromosomes of the mesenchymal stem cells is 46, and the chromosome karyotyping is a pair of chromosomes 1 to 22 and either an XX chromosome or an XY chromosome.

12. The process of separating a tissue suspension containing mesenchymal stem cells into two layers, an upper layer and a lower layer. To collect the lower layer of the two layers, and A method for culturing mesenchymal stem cells, comprising culturing the mesenchymal stem cells contained in the lower layer in a medium containing an ascorbic acid derivative, alanylglutamine, and pyridoxine, and free from extracellular matrix and scaffold.

13. The method according to claim 12, wherein the mesenchymal stem cells are synovial-derived mesenchymal stem cells.

14. The method according to claim 12 or 13, wherein the mesenchymal stem cells are of autologous origin.

15. The method according to any one of claims 12 to 14, wherein the culture medium contains the same type of serum.

16. The method according to any one of claims 12 to 15, wherein the culture medium does not contain ascorbic acid.

17. The method according to any one of claims 12 to 16, wherein the culture medium comprises one or more biotin or lipoic acid.

18. The method according to any one of claims 12 to 17, wherein mesenchymal stem cells are cultured using a multilayer flask with five or more layers.

19. A cell preparation for joint treatment comprising mesenchymal stem cells cultured by the method according to any one of claims 12 to 18.

20. A cell preparation for joint treatment according to claim 19, which is a meniscus treatment agent or an osteoarthritis treatment agent.

21. The cell preparation for joint treatment according to claim 19 or 20, wherein the mesenchymal stem cells are human mesenchymal stem cells, the number of chromosomes of the mesenchymal stem cells is 46, and the chromosome karyotyping is a pair of chromosomes 1 through 22 and either an XX chromosome or an XY chromosome.

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