Treatment for muscular dystrophy

Abstract

To provide a muscular dystrophy treatment agent that can be easily manufactured and has high therapeutic efficacy. [Solution] A cell population containing mesenchymal cells derived from amniotic tissue is included as an active ingredient in a muscular dystrophy treatment agent.

Description

[Technical Field]

[0001] This invention relates to a muscular dystrophy treatment agent containing mesenchymal cells derived from amniotic tissue. [Background technology]

[0002] Muscular dystrophy is a hereditary muscle disease characterized by degeneration and necrosis of skeletal muscle, with motor dysfunction being its primary symptom. The main types of muscular dystrophy include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, and Fukuyama congenital muscular dystrophy. DMD, in particular, is the most common and representative type of muscular dystrophy.

[0003] Regarding treatment for muscular dystrophy, there is no established cure for any type of the disease, and treatment is mostly limited to symptomatic therapy such as maintaining function through rehabilitation, assisted breathing management, and cardiac pacemakers.

[0004] Mesenchymal stem cells, which are part of stromal cells, are known as somatic stem cells found in bone marrow, adipose tissue, dental pulp, and fetal appendages such as the placenta, umbilical cord, and amniotic membrane. Somatic stem cells are particularly being applied to regenerative medicine, and in recent years, the practical application of new therapies that involve administering mesenchymal cells, such as mesenchymal stem cells, to patients to promote the regeneration and functional improvement of tissues and organs has been rapidly progressing. For example, bone marrow mesenchymal cell preparations for patients with acute graft-versus-host disease (GVHD) and spinal cord injury, and skeletal muscle blast sheets for patients with severe heart failure are being sold in Japan as regenerative medicine products.

[0005] Mesenchymal cells typically have the property of adhering to culture substrates, and when manufacturing the cell preparation, in addition to the step of adhering the cells to the culture substrate and culturing them, a step of detaching the cells that have adhered to the culture substrate is required. The bone marrow mesenchymal cells mentioned above also have the property of adhering to culture substrates, and after culturing the cells while they are adhered to the culture substrate, the cell suspension is recovered after a step of detaching the cells from the culture substrate. Methods for detaching the cells from the culture substrate include, for example, chemical means using proteolytic enzymes such as trypsin or chemicals, and physical means using instruments that physically detach cells, such as cell scrapers. For example, Patent Document 1 discloses a method of detaching mesenchymal cells by adding trypsin to a culture substrate. Patent Document 2 also discloses a method of detaching cells from a culture substrate by changing the temperature, using a culture substrate coated on its surface with a temperature-responsive polymer whose cell adhesion changes with temperature.

[0006] The amnion is a translucent membrane located on the fetal side of the placenta in pregnant women, enveloping the fetus and retaining amniotic fluid. It has been reported that mesenchymal cells are also present in the amnion (Non-Patent Literature 1). The amnion can be non-invasively collected as medical waste during childbirth, and no further invasive procedures are required for cell collection. Mesenchymal cells present in the amnion are thought to be mesenchymal stem cells because they possess properties similar to mesenchymal stem cells derived from bone marrow or fat. However, they have also been reported to have little ability to differentiate into adipocytes or vascular endothelial cells (Non-Patent Literature 2), suggesting that their cellular characteristics differ from those of bone marrow mesenchymal stem cells and adipose mesenchymal stem cells.

[0007] Regarding the treatment of DMD, the use of mesenchymal stem cells, specifically those derived from dental pulp, is being considered. Non-patent document 3 reports that intravascular administration of human dental pulp stem cells stabilized symptoms in DMD model animals. Furthermore, patent document 3 proposes a muscular dystrophy treatment agent containing pluripotent stem cell-enriched human dental pulp-derived cells as the active ingredient, and reports that administration of this agent suppressed the progression of muscle inflammation, decreased motor function, and decreased cardiac function associated with DMD in DMD model animals.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Brief Description of the Drawings

[0010] [Figure 1] It is a graph showing the results of a growth curve when amniotic mesenchymal cells (including human amniotic mesenchymal stem cells (hAMSC)) are subcultured until growth arrest in αMEM containing 10% FBS. [Figure 2] It is a graph showing the results of a growth curve when amniotic mesenchymal cells (including human amniotic mesenchymal stem cells (hAMSC)) are subcultured until growth arrest in αMEM containing 5% hPL. [Figure 3]This graph shows the results of a grip strength comparison between mdx mice (DMD model mice). The grip strength of wild-type (n=7), untreated mdx mice (n=16), mdx mice administered with amniotic mesenchymal cells (including human amniotic mesenchymal stem cells (hAMSC)) four times (n=7), and mdx mice administered with amniotic mesenchymal cells six times (n=5) were compared. In the figure, in the one-way ANOVA between wild-type and each mdx mouse, "****" indicates that P<0.0001. Also, in the one-way ANOVA between untreated mdx mice and each amniotic mesenchymal cell-treated mouse, "##" indicates P<0.01, "###" indicates P<0.001, and "ns" indicates no significant difference. [Figure 4] This graph shows the results of a comparison of circulatory function in mdx mice (DMD model mice). The left ventricular diameter shortening rate (LVFS(%)) was compared between wild-type (n=4), untreated mdx mice (n=6), and mdx mice administered four doses of amniotic mesenchymal cells (including human amniotic mesenchymal stem cells (hAMSCs)) (n=5). In the figure, "**" indicates a P<0.01 result in the t-test between wild-type and untreated mdx mice. Similarly, "#" indicates a P<0.05 result in the t-test between untreated mdx mice and mice administered amniotic mesenchymal cells. (LVFS(%)) [Overview of the project] [Problems that the invention aims to solve]

[0011] The muscular dystrophy treatment described in Patent Document 3 uses dental pulp-derived cells as its active ingredient. However, since only a small amount of dental pulp-derived cells can be collected from a single sample, their expansion culture requires considerable time and cost. Furthermore, while Patent Document 3 reports that supporting cells were used during the initial culture of dental pulp-derived cells, a process of separating the target cells from the supporting cells is required for use as a treatment agent administered to patients. Considering the practicality of the treatment agent, a more efficiently manufactured muscular dystrophy treatment agent is desired.

[0012] The present invention aims to provide a muscular dystrophy treatment agent that can be easily manufactured and has high therapeutic efficacy. [Means for solving the problem]

[0013] As a result of diligent research, the inventors discovered that by using a cell population containing mesenchymal cells derived from amniotic membrane, it is possible to improve the skeletal muscle function and cardiac function of patients suffering from muscular dystrophy, thus completing the present invention.

[0014] In other words, the following inventions are provided according to this specification. (1) A muscular dystrophy treatment agent containing a cell population including mesenchymal cells derived from amniotic tissue as an active ingredient. (2) The muscular dystrophy treatment agent according to (1), wherein the cell population has a proportion of CD324-positive mesenchymal cells of 70% or more and a proportion of CD90-positive mesenchymal cells of 90% or more. (3) The muscular dystrophy treatment agent according to (1) or (2), wherein the proportion of CD326-positive cells in the cell population is 10% or less. (4) A muscular dystrophy treatment agent according to any one of (1) to (3), wherein in the cell population, the proportion of mesenchymal cells exhibiting CD73 positivity is 80% or more, the proportion of mesenchymal cells exhibiting CD166 positivity is 80% or more, the proportion of cells exhibiting CD45 positivity is 10% or less, and the proportion of mesenchymal cells exhibiting CD105 positivity is 70% or more. (5) The muscular dystrophy treatment agent according to any one of (1) to (4), wherein the cell population is obtained from a living organism different from the subject to which the agent is administered. (6) The muscular dystrophy treatment agent according to any one of (1) to (4), wherein the cell population is obtained from a living organism of the same species as the subject to administration. (7) A muscular dystrophy treatment agent described in any of (1) to (6), which is administered to humans. (8) A muscular dystrophy treatment agent according to any one of (1) to (7), wherein the cell population is of human origin. (9) The muscular dystrophy treatment agent according to any one of (1) to (8), wherein the mesenchymal cells have no or low ability to differentiate into osteoblasts and adipocytes. (10) The mesenchymal cells have an anti-inflammatory effect, and the muscular dystrophy treatment agent according to any one of (1) to (9). (11) The muscular dystrophy treatment agent according to (1) to (10), wherein the cell population is a population of cells cultured using a medium containing platelet lysate. (12) A muscular dystrophy treatment agent described in any of (1) to (11), which is administered intravenously. (13) A muscular dystrophy treatment agent described in any of (1) to (12) that provides therapeutic effects for six months or more after administration. (14) A muscular dystrophy treatment agent described in any of (1) to (13), used for the treatment of Duchenne muscular dystrophy. (15) A method for producing a muscular dystrophy treatment agent, comprising the steps of (a) preparing a cell population containing mesenchymal cells from amniotic tissue; and (b) culturing the cell population from (a). (16) The method according to (15), wherein a culture medium containing platelet lysate is used for the culture in (b) above. This specification includes the disclosures of Japanese Patent Application No. 2021-059235, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0015] According to the present invention, it is possible to provide a muscular dystrophy treatment agent that can be manufactured more easily than conventional agents and has a higher therapeutic effect. [Modes for carrying out the invention]

[0016] [1] Explanation of terms In this specification, "fetal appendages" refers to the amniotic membrane, placenta, umbilical cord, and amniotic fluid. Furthermore, the "amniotic membrane" is the gestational sac containing the amniotic fluid of the fetus, and consists of the amnion, chorion, and decidua from the inside out. Of these, the amnion originates from the fetus. The "amnion" refers to the transparent, vascularly sparse membrane that forms the innermost layer of the amniotic membrane. The inner layer of the amnion (also called the epithelial cell layer) is covered with a single layer of epithelial cells with secretory function and secretes amniotic fluid, while the outer layer of the amnion (also called the extracellular matrix layer, which corresponds to the stroma) contains mesenchymal cells.

[0017] In this specification, "mesenchymal cells" include "mesenchymal stem cells (MSCs)" and refer to cells that meet the following definitions. i) The culture exhibits adhesion to plastic under standard culture conditions. Standard culture media are culture media prepared by adding serum, serum substitute reagents, or growth factors (e.g., human platelet lysate, which is a serum substitute reagent) to a basal medium (e.g., αMEM medium). ii) Surface antigens CD73 and CD90 are positive, while CD45 and CD326 are negative.

[0018] In this specification, "mesenchymal stem cells" are used interchangeably with "mesenchymal stromal cells." In this specification, "mesenchymal stem cells" may be referred to as "MSCs."

[0019] In this specification, the "proportion of mesenchymal cells that are positive for a given surface antigen" refers to the proportion of cells that are positive for surface antigen expression. In this specification, the proportion of cells that are positive for a given surface antigen may be referred to as the "positive rate," and the proportion of cells that are negative for a given surface antigen may be referred to as the "negative rate."

[0020] In this specification, "cell population containing mesenchymal cells" is not particularly limited as long as it contains at least mesenchymal cells, and may also contain other cells. Mesenchymal cells may be isolated from only one tissue or a mixture of cells isolated from multiple tissues. Furthermore, the form of "cell population containing mesenchymal cells" is not particularly limited and may include, for example, cell pellets, cell aggregates, cell sheets, cell suspensions, cell suspensions, and frozen versions thereof.

[0021] The surface antigen "CD324" refers to differentiation cluster 324 and is a protein also known as epithelial cadherin (E-cadherin).

[0022] The surface antigen "CD90" refers to differentiation cluster 90 and is a protein also known as Thy-1.

[0023] The surface antigen "CD326" refers to differentiation cluster 326 and is a protein also known as EpCAM.

[0024] The surface antigen "CD73" refers to differentiation cluster 73 and is a protein also known as 5-nucleotidase or ecto-5'-nucleotidase.

[0025] The surface antigen "CD166" refers to differentiation cluster 166 and is a protein also known as activated leukocyte cell adhesion molecule (ALCAM).

[0026] The surface antigen "CD105" refers to differentiation cluster 105 and is a protein also known as Endoglin.

[0027] The surface antigen "CD45" refers to differentiation cluster 45 and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type, C) or LCA (Leukocyte common antigen).

[0028] The surface antigen "CD34" refers to differentiation cluster 34 and is a protein also known as hematopoietic progenitor cell antigen CD34.

[0029] [2] Treatment for muscular dystrophy The present invention is characterized by containing a cell population including mesenchymal cells derived from amniotic tissue as an active ingredient for the treatment of muscular dystrophy.

[0030] The target population for the muscular dystrophy treatment agent of the present invention is typically humans, but other animals may also be administered. Other animals include mammals such as dogs, cats, cattle, horses, pigs, sheep, monkeys, and ferrets, and birds such as chickens. The preferred target population is humans. The following description of the muscular dystrophy treatment agent of the present invention will focus on embodiments administered to humans, but this invention is not intended to limit the target population to humans.

[0031] [2-1] Cell population containing mesenchymal cells derived from amniotic tissue In the muscular dystrophy treatment agent of the present invention (hereinafter also referred to as the "treatment agent"), the ratio of mesenchymal cells included in the "cell population including mesenchymal cells" is not particularly limited, but may be 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0032] Furthermore, the proportion of other cells in the cell population including the mesenchymal cells may be 30% or less, 20% or less, 10% or less, or 5% or less. The other cells are not particularly limited as long as they are not mesenchymal cells, but examples include hematopoietic cells such as lymphocytes, granulocytes, and erythrocytes.

[0033] The cell population containing mesenchymal cells used in the therapeutic agent of the present invention is not particularly limited as long as it contains cells that satisfy the above definitions i) and ii), but it is preferable that it be a cell population prepared to have the following characteristics (a) and (b). (a) In the cell population, the proportion of mesenchymal cells that are positive for CD324 is 70% or more, (b) In the cell population, the proportion of mesenchymal cells that are positive for CD90 is 90% or more. By possessing the aforementioned characteristics, the cell population, including mesenchymal cells, can spontaneously detach from the substrate after culture. This makes it possible to efficiently obtain cells after culture without using enzymes or special equipment, as described in Patent Document 1 or 2.

[0034] In the aforementioned cell population, the proportion of mesenchymal cells exhibiting CD324 positivity may more preferably be 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0035] In the aforementioned cell population, the proportion of CD90-positive mesenchymal cells may more preferably be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0036] In one or more embodiments of the therapeutic agent of the present invention, the cell population preferably has a proportion of CD326-positive cells of 10% or less.

[0037] In the aforementioned cell population, the proportion of CD326-positive cells may more preferably be 5% or less (negative rate of 95% or more), 4% or less (negative rate of 96% or more), 3% or less (negative rate of 97% or more), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or 0% (negative rate of 100%).

[0038] According to one aspect of the present invention, a cell population including mesenchymal cells provided by the present invention preferably satisfies one or more of the following conditions: the proportion of CD73-positive mesenchymal cells is 80% or more; the proportion of CD166-positive mesenchymal cells is 80% or more; the proportion of CD105-positive mesenchymal cells is 70% or more; the proportion of CD45-positive cells is 10% or less; and the proportion of CD34-positive cells is 10% or less.

[0039] The proportion of CD73-positive mesenchymal cells in the aforementioned cell population may more preferably be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0040] The proportion of mesenchymal cells exhibiting CD166 positivity in the aforementioned cell population may more preferably be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0041] The proportion of mesenchymal cells exhibiting CD105 positivity in the aforementioned cell population may more preferably be 74% or more, 75% or more, 80% or more, 85% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0042] The proportion of CD45-positive cells in the aforementioned cell population may more preferably be 5% or less (negative rate of 95% or more), 4% or less (negative rate of 96% or more), 3% or less (negative rate of 97% or more), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or even 0% (negative rate of 100%).

[0043] The proportion of CD34-positive cells in the aforementioned cell population may more preferably be 5% or less (negative rate of 95% or more), 4% or less (negative rate of 96% or more), 3% or less (negative rate of 97% or more), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or 0% (negative rate of 100%).

[0044] Here, cells or mesenchymal cells exhibiting CD324-positive, CD90-positive, CD326-positive, CD73-positive, CD166-positive, CD105-positive, CD45-positive, and CD34-positive status refer to cells or mesenchymal cells that are positive for the expression of CD324, CD90, CD326, CD73, CD166, CD105, CD45, and CD34, respectively.

[0045] The cell population used in the therapeutic agent of the present invention can be detected by any detection method known in the art, such as CD324, CD90, CD326, CD73, CD166, CD105, CD45, or CD34. Examples of methods for detecting the expression marker include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, if cells emitting stronger fluorescence than the negative control (isotype control) are detected, those cells are determined to be "positive" for the marker. Any fluorescently labeled antibody known in the art can be used, such as, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. In cell staining, if cells that are stained or fluorescein are observed under a microscope, those cells are determined to be "positive" for the marker. Cell staining may be immunohistochemistry using antibodies or non-immunohistochemistry without antibodies. In this specification, expression markers and surface antigens are synonymous and can be used interchangeably.

[0046] The cell population containing mesenchymal cells used in the therapeutic agent of the present invention is a cell population containing mesenchymal cells isolated from amniotic tissue. Mesenchymal cells isolated from amniotic tissue (hereinafter also referred to as "amniotic mesenchymal cells") can be isolated in large numbers from the raw tissue and have high proliferative capacity, allowing a predetermined amount of cells to be obtained in a short period of time. Therefore, amniotic mesenchymal cells can be stably obtained as high-quality cells with a low passage count, and since supporting cells are not necessarily required during culture, they can be manufactured at a relatively low cost. As amniotic mesenchymal cells, those obtained from a different organism than the target of administration, i.e., allogeneic amniotic mesenchymal cells, can be used.

[0047] The cell population containing the aforementioned mesenchymal cells is preferably a cell population having anti-inflammatory activity. Amniotic mesenchymal stem cells contained in amniotic mesenchymal cells are known to have high anti-inflammatory activity (see, for example, Yamahara K, et al., Pros One, 9(2) e88319 (2014)), and in the therapeutic agent of the present invention, the method for collecting and sorting the cell population is preferably carried out under conditions that maintain its anti-inflammatory activity.

[0048] The amniotic mesenchymal cells may be derived from any mammal, for example, rodents such as mice, rats, and hamsters; primates such as humans, gorillas, and chimpanzees; and domestic or pet mammals such as dogs, cats, rabbits, cattle, horses, sheep, and goats, but it is particularly preferable that they be derived from the same animal species as the target of administration. If the target of administration is a human, it is preferable that the amniotic mesenchymal cells are derived from humans.

[0049] The amniotic mesenchymal cells described above are preferably those that have little or no ability to differentiate into osteoblasts and adipocytes. In this specification, mesenchymal cells with little or no ability to differentiate into osteoblasts refer to mesenchymal cells in which, when cells cultured for 21 days in any known calcification-inducing medium (for example, commercially available media such as StemMACS® OsteoDiff Media, MesenCult® Osteogenic Differentiation Kit, or StemPro® Osteogenesis Differentiation Kit can be used), the proportion of stained cells in the cell population under the microscope does not exceed 40% (most preferably 0%) when stained with alizarin red. There have been reports that muscle symptoms stabilized in muscular dystrophy model animals injected with MSCs derived from dental pulp cells (Patent Document 3 and Non-Patent Document 3), but since the amniotic mesenchymal cells in this invention have little or no ability to differentiate into osteoblasts, their differentiation ability is significantly different from that of dental pulp cells, resulting in differences in cellular characteristics.

[0050] Furthermore, in this specification, mesenchymal cells with no or low ability to differentiate into adipocytes refer to mesenchymal cells in which, when cells cultured for 21 days or more in any known adipocyte differentiation induction medium (for example, commercially available media such as StemMACS® AdipoDiff Media, MesenCult® Adipogenic Differentiation Kit, and StemPro® Adipogenesis Differentiation Kit can be used), the proportion of stained cells in the cell population under the microscope does not exceed 20% (most preferably 0%) when stained with Oil Red O. More preferably, mesenchymal cells with no or low ability to differentiate into adipocytes refer to mesenchymal cells in which, when cultured using two or three of the adipocyte differentiation induction media from StemMACS® AdipoDiff Media, MesenCult® Adipogenic Differentiation Kit, and StemPro® Adipogenesis Differentiation Kit, in any case, when cells cultured for 21 days or more are stained with Oil Red O, the proportion of stained cells in the cell population under the microscope does not exceed 20%.

[0051] [2-2] Recovery of cell populations including mesenchymal cells A method for recovering a cell population containing mesenchymal cells from a sample may include a step of enzymatically treating the amnion or fetal appendages containing the amnion and / or adhering them to a culture vessel to obtain a cell population containing mesenchymal cells. The recovered cell population may contain other miscellaneous cells as long as it contains amnion-mesenchymal cells. When obtaining cells from tissue, blood cells may be mixed in from blood attached to the tissue.

[0052] The primary cells of the aforementioned cell population are preferably cells obtained by treating the amnion with at least collagenase. The amnion consists of an epithelial cell layer and an extracellular matrix layer, and amnional mesenchymal cells are included in the latter. The enzymatic treatment of the amnion is preferably performed with an enzyme (or a combination thereof) that can release mesenchymal cells contained in the extracellular matrix layer of the amnion without degrading the epithelial cell layer. Such enzymes are not particularly limited, but examples include collagenase and / or metalloproteinases. Examples of metalloproteinases include thermolysin and / or dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids, but are not particularly limited.

[0053] The activity concentration of collagenase is preferably 50 PU / ml or higher, more preferably 100 PU / ml or higher, even more preferably 200 PU / ml or higher, even more preferably 300 PU / ml or higher, and even more preferably 400 PU / ml or higher. The activity concentration of collagenase is not particularly limited, but for example, it may be 1000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, or 500 PU / ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 ug of FITC-collagen in 1 minute at pH 7.5 and 30°C.

[0054] The activity concentration of the metalloproteinase (e.g., thermolysin and / or dispase) is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, even more preferably 200 PU / ml or more, even more preferably 300 PU / ml or more, and even more preferably 400 PU / ml or more. Alternatively, the activity concentration of the metalloproteinase is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, even more preferably 800 PU / ml or less, even more preferably 700 PU / ml or less, even more preferably 600 PU / ml or less, and even more preferably 500 PU / ml or less. Here, in the embodiment using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that releases an amino acid equivalent to 1 ug of tyrosine per minute from lactate casein at pH 7.5 and 30°C. Within the above enzyme concentration range, mesenchymal cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of the epithelial cell layer contained in the fetal appendage's epithelial cell layer. The preferred combination of collagenase and / or metalloproteinase concentrations can be determined by microscopic observation of fetal appendages after enzyme treatment or by flow cytometry of the acquired cells.

[0055] From the viewpoint of efficiently recovering viable cells, it is preferable to simultaneously process fetal appendages in a single step using a combination of collagenase and metalloproteinase. In this case, thermolysin and / or dispase can be used as the metalloproteinase, but are not limited to these. Mesenchymal cells can be easily obtained by processing fetal appendages only once with an enzyme solution containing collagenase and metalloproteinase. Furthermore, simultaneous processing reduces the risk of contamination by bacteria, viruses, etc.

[0056] Enzymatic treatment of the amniotic membrane is preferably carried out by immersing the amniotic membrane, which has been washed with a washing solution such as physiological saline or Hanks equilibrium salt solution, in an enzyme solution and treating it while stirring with a stirring device. As such a stirring device, a stirrer or shaker can be used, but is not limited to these, from the viewpoint of efficiently releasing mesenchymal cells contained in the extracellular matrix layer of the fetal appendages. The stirring speed is not particularly limited, but when using a stirrer or shaker, for example, it is 5 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, or 50 rpm or more. The stirring speed is not particularly limited, but when using a stirrer or shaker, for example, it is 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less. The enzyme treatment time is not particularly limited, but for example, it is 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, or 90 minutes or more. Furthermore, the enzyme treatment time is not particularly limited, but for example, it may be 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, or 100 minutes or less. The enzyme treatment temperature is not particularly limited, but for example, it may be 15°C or higher, 16°C or higher, 17°C or higher, 18°C ​​or higher, 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, or 36°C or higher. Furthermore, the enzyme treatment temperature is not particularly limited, but for example, it may be 40°C or lower, 39°C or lower, 38°C or lower, or 37°C or lower.

[0057] If desired, the free mesenchymal cells can be separated and / or recovered from the enzyme solution containing the free mesenchymal cells by known methods such as filtering, centrifugation, hollow fiber separation membranes, or cell sorters. Preferably, the enzyme solution containing the free mesenchymal cells is filtered by a filter. In the embodiment in which the enzyme solution is filtered by a filter, only the free cells pass through the filter, and the undegraded epithelial cell layer remains on the filter without passing through it. This not only allows for easy separation and / or recovery of the free mesenchymal cells, but also reduces the risk of contamination by bacteria, viruses, etc. The filter is not particularly limited, but for example, a mesh filter can be used. The pore size (mesh size) of the mesh filter is not particularly limited, but for example, it may be 40 μm or larger, 50 μm or larger, 60 μm or larger, 70 μm or larger, 80 μm or larger, or 90 μm or larger. Furthermore, the pore size of the mesh filter is not particularly limited, but for example, it may be 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, or 100 μm or less. The filtration rate is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzyme solution containing mesenchymal cells can be filtered by gravity, thereby preventing a decrease in cell viability.

[0058] Nylon is preferred as the material for the mesh filter. Tubes containing 40μm, 70μm, 95μm, or 100μm nylon mesh filters, such as the Falcon cell strainer commonly used for research purposes, are available. Medical mesh cloths (nylon and polyester) used in hemodialysis and other applications can also be used. Furthermore, arterial filters (polyester mesh filters, pore size: 40μm to 120μm) used during extracorporeal circulation are also available. Other materials, such as stainless steel mesh filters, can also be used.

[0059] When filtering mesenchymal cells, gravity (free fall) is preferred. Forced filtering using a pump or other means is also possible, but to avoid damaging the cells, it is desirable to use the lowest possible pressure.

[0060] The filtered mesenchymal cells can be recovered by centrifugation after diluting the filtrate with twice the volume or more of culture medium or equilibrium salt buffer. Suitable equilibrium salt buffers include, but are not limited to, Dulbecco's phosphate buffer (DPBS), Earl's equilibrium salt solution (EBSS), Hanks' equilibrium salt solution (HBSS), and phosphate buffer (PBS).

[0061] [2-3] Method for selecting cell populations The cell population used in the therapeutic agent of the present invention may be prepared by a selection process of a cell population having the following characteristics (a) and (b). (a) The proportion of mesenchymal cells exhibiting CD324 positivity is 70% or more, (b) The proportion of mesenchymal cells that are CD90 positive is 90% or more.

[0062] Means for selecting cell populations based on the aforementioned characteristics include physical means such as FACS, cell sorting, and separation using magnetic beads, as well as chemical means of purifying the cell population, including mesenchymal cells, by eliminating cells that do not meet the criteria under appropriate culture conditions. However, the means are not particularly limited and can be appropriately selected depending on the culture conditions, etc. In the selection process, it is preferable to carry out the culture in a medium containing platelet lysate.

[0063] The timing for selecting cell populations having the characteristics of (a) and (b) above is not particularly limited, but examples include before culturing, during culturing, after culturing, before harvesting the cell population, after harvesting the cell population, before creating a cryopreservation stock of cells, and after thawing a cryopreservation stock of cells.

[0064] The procedure for selecting cell populations having the characteristics of (a) and (b) described above will be explained in more detail. For example, one method is to seed a cell population containing mesenchymal cells onto a culture substrate and culture it on the culture substrate, thereby positively selecting mesenchymal cells that are CD324-positive and CD90-positive, and then detaching and collecting the cell population in which the proportion of CD324-positive mesenchymal cells is 70% or more and the proportion of CD90-positive mesenchymal cells is 90% or more. At this time, culture may be performed using a culture substrate coated with anti-CD324 antibody or anti-CD90 antibody. The timing of selecting CD324-positive and CD90-positive mesenchymal cells from a cell population containing mesenchymal cells by culture is not particularly limited and can be done at any subculturing stage, however, it is preferable to select CD324-positive and CD90-positive mesenchymal cells from a cell population containing mesenchymal cells in the primary culture.

[0065] Furthermore, by using cell separation methods such as flow cytometry or magnetic beads, mesenchymal cells that are both CD324-positive and CD90-positive can be selected from a cell population that includes mesenchymal cells.

[0066] For further selection of the cell population, it is preferable to select and separate a cell population in which the proportion of CD326-positive cells is 10% or less. Furthermore, it is preferable to select and separate a cell population that satisfies one or more of the following conditions: the proportion of CD73-positive mesenchymal cells is 80% or more; the proportion of CD166-positive mesenchymal cells is 80% or more; the proportion of CD105-positive mesenchymal cells is 70% or more; the proportion of CD45-positive cells is 10% or less; and the proportion of CD34-positive cells is 10% or less. The selected cell population may be further cultured by the means described above.

[0067] [2-4] Methods for culturing mesenchymal cells The cell population, including mesenchymal cells, recovered by the method described above can be proliferated by culturing. The seeding density of the cell population in culture is, for example, 500 to 10,000 cells / cm³. 2include. The density at the time of seeding the cell population is more preferably 500 cells / cm 2 or more, 1,000 cells / cm 2 or more, 2,000 cells / cm 2 or more, 3,000 cells / cm 2 or more, 4,000 cells / cm 2 or more, 5,000 cells / cm 2 or more. The density at the time of seeding the cell population is more preferably 10,000 cells / cm 2 or less, 9,000 cells / cm 2 or less, 8,000 cells / cm 2 or less, 7,000 cells / cm 2 or less.

[0068] The above culture method may include a subculture step, or may include a step of repeating culturing multiple times under different culture conditions. As the culture period of one culture, for example, 4 to 10 days can be mentioned, and more specifically, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days can be mentioned.

[0069] The medium used for the above culture can be prepared by using any liquid medium for animal cell culture as a basal medium and appropriately adding other components (serum, serum replacement reagent, growth factor, etc.) as necessary.

[0070] As basal media, you may use, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)).

[0071] Other components include, for example, albumin, serum, serum substitute reagents, or growth factors. In embodiments where growth factors are added, the mixture may be prepared by adding a reagent for stabilizing the growth factor in the culture medium (such as heparin) to the growth factor and then adding the reagent to the culture medium, or by pre-stabilizing the growth factor with a gel or polysaccharide and then adding the stabilized growth factor to the basal culture medium. In the case of albumin, a concentration of more than 0.05% and 5% or less is preferred. In the case of serum, a concentration of 5% or more is preferred.

[0072] The inventors have found that a cell population including mesenchymal cells cultured in a medium containing platelet lysate (PL), particularly human PL (hPL), can be subcultured for a longer period compared to mesenchymal cells cultured in a medium containing FBS instead of PL. Therefore, it is preferable that the other component includes PL, particularly hPL. The lower limit of the hPL concentration in the medium can be, for example, 1% by weight or more, 2% by weight or more, or 3% by weight or more as a final concentration. The upper limit of the hPL concentration in the medium can be, for example, 20% by weight or less, 10% by weight or less, or 7% by weight or less.

[0073] The culture medium used for the above-mentioned culture may be a commercially available serum-free medium. Examples include, but are not limited to, STK1 or STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), and Corning stemgro Human Mesenchymal Stem Cell Medium (Corning).

[0074] Cell populations including mesenchymal cells can be cultured using, for example, the following procedure: First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Next, the cells are seeded in a plastic culture vessel and cultured in culture medium at a CO2 concentration of 3% to 5% and a temperature of 37°C until the confluence rate is 95% or less. Examples of the culture medium include, but are not limited to, αMEM, M199, or media based on these. Cells obtained by the above culture method are cells that have been cultured once.

[0075] The cells cultured once as described above can be further subcultured as follows: First, the cells cultured once are treated with ethylenediaminetetraacetic acid (EDTA) and then with trypsin to detach them from the plastic culture vessel. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Finally, the cells are seeded in a plastic culture vessel and cultured in culture medium at a CO2 concentration of 3% to 5% and a temperature of 37°C until the confluence rate is 95% or less. Examples of the culture medium include, but are not limited to, αMEM, M199, or media based on these. Cells obtained by the above subculturing and culture are cells that have been subculturised once. By performing similar subculturing and culture, cells that have been subculturised N times can be obtained (where N is an integer greater than or equal to 1). From the viewpoint of mass production of cells, the lower limit of the number of passages N is, for example, 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, even more preferably 6 or more, even more preferably 7 or more, even more preferably 8 or more, even more preferably 9 or more, even more preferably 10 or more, even more preferably 11 or more, even more preferably 12 or more, even more preferably 13 or more, even more preferably 14 or more, even more preferably 15 or more, even more preferably 16 or more, even more preferably 17 or more, even more preferably 18 or more, even more preferably 19 or more, even more preferably 20 or more, and even more preferably 25 or more. Furthermore, from the viewpoint of suppressing cell aging, the upper limit of the number of passages N is preferably, for example, 50 or less, 45 or less, 40 or less, 35 or less, and 30 or less.

[0076] In the culture process described above, mesenchymal cells can be cultured without growth arrest for at least 40 days, more preferably 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, or 110 days after the start of in vitro culture.

[0077] In the culture process described above, mesenchymal cells can be cultured for at least 10, more preferably 15, 20, 25, 30, 35, 40, 45, or 50 doubling cycles after the start of in vitro culture.

[0078] The doubling count is the number of times a cell divides during a certain culture period, [log 10 (Number of cells at the end of culture) - log 10 (Number of cells at the start of culture)] / log 10 It is calculated using formula (2). If subculturing is performed, the total number of doublings is calculated by first calculating the number of doublings for each subculturing using the formula above and then accumulating them.

[0079] [2-5] Methods for preserving cell populations including mesenchymal cells Cell populations containing mesenchymal cells can be cryopreserved. When the cell population is cryopreserved, it may be separated, recovered, and / or cultured after thawing, as needed. Alternatively, the cell population may be used as is after thawing.

[0080] The means for cryopreserving the cell population are not particularly limited, but include, for example, a programmable freezer, a deep freezer, or immersion in liquid nitrogen. The freezing temperature is preferably -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, -70°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -110°C or lower, -120°C or lower, -130°C or lower, -140°C or lower, -150°C or lower, -160°C or lower, -170°C or lower, -180°C or lower, -190°C or lower, or -196°C (liquid nitrogen temperature) or lower. Preferred freezing rates are, for example, 1°C / min, 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min, 10°C / min, 11°C / min, 12°C / min, 13°C / min, 14°C / min, or 15°C / min. When a programmable freezer is used as the freezing method, for example, the temperature can be lowered to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of 1°C / min to 2°C / min, and then further lowered to a temperature between -100°C and -80°C (e.g., -90°C) at a freezing rate of 9°C / min to 11°C / min (e.g., 10°C / min).

[0081] When freezing using the freezing method described above, the cell population may be frozen in any storage container. Examples of such storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, and infusion bags.

[0082] From the viewpoint of increasing cell viability, the cryopreservation solution preferably contains a predetermined concentration of albumin greater than 0% by mass. Preferred concentrations of albumin are, for example, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, or 8% by mass or more. Alternatively, preferred concentrations of albumin are, for example, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

[0083] [2-6] Therapeutic agents The present invention provides a muscular dystrophy treatment agent, which is a pharmaceutical composition used for the treatment of muscular dystrophy, comprising a cell population including mesenchymal cells derived from the amniotic tissue described above as an active ingredient.

[0084] The therapeutic agent of the present invention is not particularly limited to muscular dystrophy, and may be any of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, or Fukuyama congenital muscular dystrophy. It is particularly suitable for the treatment of DMD. The therapeutic agent of the present invention is used to restore the function of the target muscles, especially skeletal muscles, that has been reduced due to muscular dystrophy. Alternatively, it is used to prevent the decline of muscle function in patients with muscular dystrophy.

[0085] Furthermore, the therapeutic agent of the present invention can also be used for symptoms of muscle weakness in patients other than those with muscular dystrophy, such as sarcopenia and frailty.

[0086] The therapeutic agent of the present invention preferably has a therapeutic effect that lasts for 6 months or more, more preferably 12 months or more.

[0087] The therapeutic agent of the present invention may be administered in mixture with other mesenchymal cells.

[0088] The dosage of the therapeutic agent of the present invention is a dosage that, when administered to a patient or subject, results in a cell volume that provides a therapeutic effect on the disease compared to patients or subjects who do not receive the agent. The specific dosage can be appropriately determined depending on the form of administration, method of administration, purpose of use, age of the patient or subject, symptoms, and the amount of adipose tissue to be engrafted. The dosage is not particularly limited, but for example, 10 per administration 5 More than 10 pieces 6 One or more or 10 7 It is 10 or more. Also, the dosage is not particularly limited, but for example, 10 per dose. 10Less than or equal to 10 9 10 or fewer or 10 8 It is less than or equal to one.

[0089] The method of administering the therapeutic agent of the present invention is not particularly limited, but examples include systemic administration by intravascular injection (e.g., venous injection), direct injection to a local area, or direct transplantation to a local area.

[0090] The therapeutic agent of the present invention can also be used as an injectable formulation, a transplantable formulation in the form of cell aggregates or sheet-like structures, or a gel formulation mixed with any gel for the purpose of treating other diseases.

[0091] The therapeutic agent of the present invention may be used immediately after culturing, but it is preferable to store it in a frozen state until immediately before use. The period for which it can be frozen before administration is preferably one month or more, more preferably six months or more, and even more preferably one year or more. The therapeutic agent of the present invention may contain any component used in the treatment of humans or any component used in the cryopreservation of cells. Examples of such components include, but are not limited to, salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, and culture medium components (e.g., components contained in RPMI1640 medium, etc.). For example, it is preferable to contain dimethyl sulfoxide, hydroxyethyl starch, and human albumin, and it is even more preferable to contain 5-10% by mass of dimethyl sulfoxide, 4-10% by mass of hydroxyethyl starch, and 5% by mass or less of human albumin.

[0092] The therapeutic agent of the present invention may be a cell population including mesenchymal cells diluted with an intravenous fluid preparation used as a pharmaceutically acceptable medium. The term "intravenous fluid preparation (pharmaceutically acceptable medium)" as used herein is not particularly limited as long as it is a solution used in the treatment of humans, but examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, initiation solution (Solution 1), dehydration replacement solution (Solution 2), maintenance intravenous fluid (Solution 3), postoperative recovery solution (Solution 4), etc.

[0093] [3] Method for manufacturing a drug for treating muscular dystrophy The present invention provides a method for producing a muscular dystrophy treatment agent (hereinafter also referred to as the "production method"), characterized by comprising: (a) a step of preparing a cell population including mesenchymal cells from amniotic tissue; and (b) a step of culturing the cell population from (a).

[0094] [3-1] Step of preparing a cell population containing mesenchymal cells from amniotic tissue (Step (a)) The manufacturing method of the present invention includes a step of preparing a cell population containing mesenchymal cells from amniotic tissue (hereinafter also referred to as "step (a)"). In the manufacturing method of the present invention, the "step of preparing a cell population" includes a step of recovering a cell population containing mesenchymal cells from amniotic tissue, and optionally includes a step of selecting the cell population. Unless otherwise specified, the conditions in step (a) are the same as those described in the sections "[2-2] Method for recovering a cell population containing mesenchymal cells" and "[2-3] Method for selecting a cell population" above.

[0095] [3-2] Steps to culture the cell population The manufacturing method of the present invention includes a step of culturing the cell population of (a) (hereinafter also referred to as "step (b)"). The culture medium used in step (b) is not particularly limited, but the use of a medium containing platelet lysate is particularly preferred. The other conditions in step (b) are the same as those described in the section "[2-4] Method for culturing a cell population containing mesenchymal cells" above, unless otherwise specified.

[0096] [3-3] Other processes The manufacturing method of the present invention may further include a cell population preservation step, a formulation step, etc. The detailed conditions of each step are the same as those described in the sections "[2-5] Method for preserving a cell population containing mesenchymal cells" and "[2-6] Therapeutic agent" above, unless otherwise specified.

[0097] [4] How to treat muscular dystrophy This specification provides a method for treating a target muscular dystrophy. The method for treating a target muscular dystrophy as described herein is characterized by comprising the step of administering a muscular dystrophy therapeutic agent containing a cell population including amniotic mesenchymal cells as an active ingredient to a target.

[0098] The detailed conditions for muscular dystrophy treatment agents and methods for manufacturing muscular dystrophy treatment agents used in methods for treating muscular dystrophy are as described in sections "[2] Muscular Dystrophy Treatment Agents" and "[3] Methods for Manufacturing Muscular Dystrophy Treatment Agents," unless otherwise specified. [Examples]

[0099] <Example 1: FBS culture of amniotic mesenchymal cells> (Step 1-1: Amniotic membrane harvesting) The amniotic membrane and placenta, which are fetal appendages, were aseptically collected from a pregnant woman (donor #1) in a case of elective cesarean section for whom informed consent had been obtained. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amnion was manually separated from the cut end of the amniotic membrane. The amnion was washed with Hanks equilibrium salt solution (Ca·Mg-free) to remove any attached blood and blood clots.

[0100] (Steps 1-2: Enzymatic treatment of amniotic membrane and collection of mesenchymal cells) The amnion, containing both epithelial and mesenchymal cell layers, was enzymatically treated by immersing it in Hanks equilibrium salt solution (containing Ca·Mg) with 240 PU / mL collagenase and 200 PU / mL dispase I, and shaking it at 50 rpm at 37°C for 90 minutes. After enzymatic treatment, the solution was filtered through a 95 μm nylon mesh to remove undigested amnional material, and a cell suspension containing mesenchymal cells was recovered.

[0101] (Steps 1-3: Culture of mesenchymal cells) The cell population containing mesenchymal cells obtained in "Step 1-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" described above was seeded in a CellSTACK® culture vessel (manufactured by Corning). The seeding density was 6,000 cells / cm².2 Cells were seeded at the specified density. After seeding, the cells were adherently cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) until subconfluent. After culturing, 15 mL of TrypLE Select was added to each CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained adhered to the CellSTACK® in the culture vessel without detaching. Therefore, the cells were incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here was the 0th passage. Subsequently, 1 / 5 of the above cell population was seeded in CellSTACK® of the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF. The culture medium was changed every 2-4 days. When subconfluence was reached, 15 mL of TrypLE Select was added to each CellSTACK® stack, and incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained attached to the CellSTACK® in the culture vessel without detaching. Therefore, incubation was continued for an additional 5 minutes (total of 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the first passage.

[0102] Subsequently, the cell concentration increased to 2 × 10 7 RPMI1640 was added to achieve a cell / mL ratio. An equal volume of CP-1(registered trademark) solution (CP-1(registered trademark): 25% human serum albumin = 34:16 ratio) was added to this solution, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 15,000-18,000 cells / cm³. 2At the specified density, the cell population of the first passage was seeded onto CellSTACK® and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained attached to the CellSTACK® in the culture vessel without detaching. Therefore, the cells were incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the second passage.

[0103] Subsequently, 1 / 5 of the aforementioned cell population was seeded into CellSTACK® on the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF. The culture medium was changed every 2-4 days. When subconfluence was reached, 15 mL of TrypLE Select was added per CellSTACK® stack, and incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained attached to the CellSTACK® in the culture vessel without detaching. Therefore, incubation was continued for an additional 5 minutes (total of 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the 3rd passage.

[0104] For the aforementioned cell population, the cell concentration is 4 × 10 6 RPMI1640 was added to achieve a cell / mL ratio. An equal volume of CP-1(registered trademark) solution (CP-1(registered trademark): 25% human serum albumin = 34:16 ratio) was added, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 6,000 cells / cm³.2 The cell population of the third passage was seeded on CellSTACK® at a density of , and adherent culture was carried out in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK® stack, and the culture was incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained adhered to the CellSTACK® in the culture vessel without detaching. Therefore, the culture was incubated for an additional 5 minutes (total of 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the fourth passage.

[0105] Subsequently, 1 / 5 of the cell population was seeded into CellSTACK® on the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF. The culture medium was changed every 2-4 days. When subconfluence was reached, 15 mL of TrypLE Select was added per CellSTACK® stack, and incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained attached to the CellSTACK® in the culture vessel without detaching. Therefore, incubation was continued for an additional 5 minutes (total of 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here was the 5th subgeneration. Subsequently, the cell concentration was 4 × 10⁶ 6 RPMI1640 was added to achieve a cell / mL concentration. An equal volume of CP-1(registered trademark) solution (CP-1(registered trademark): 25% human serum albumin = 34:16 ratio) was added to this solution, and 1 mL portions were transferred to cryovials and slowly frozen to -80°C, after which they were stored under liquid nitrogen.

[0106] (Steps 1-4: Surface antigen analysis of mesenchymal cells) For a cell population of 5 passages cultured using the above culture method, various surface antigens (positive rates for CD324, CD73, CD90, CD105, CD166, CD45, and CD326) were analyzed using a flow cytometer. The results showed that the positive rate for CD324 was less than 70% (specifically 33%), the positive rate for CD105 was 70% or higher (specifically 93%), and the positive rates for CD73, CD90, and CD166 were all 90% or higher (specifically CD73: 99%, CD90: 93%, CD166: 97%). The negative rates for CD45 and CD326 were both 95% or higher (specifically CD45: 100%, CD326: 100%). From these results, it was found that the cell population cultured using the above culture method is a cell population that includes mesenchymal cells. Furthermore, it was found that the cell population at the 5th passage of Example 1 met the condition that the proportion of CD90-positive mesenchymal cells was 90% or more, but did not meet the condition that the proportion of CD324-positive mesenchymal cells was 70% or more.

[0107] For this measurement, REA Control(S)APC (Miltenyi Biotec, clone: ​​REA293, model number: 130-113-434) was used as the isotype control antibody. CD324-APC, Human (Miltenyi Biotec, clone: ​​REA811, model number: 130-111-840) was used as the antibody against the CD324 antigen, CD73-APC, Human (Miltenyi Biotec, clone: ​​REA804, model number: 130-111-909) was used as the antibody against the CD73 antigen, CD90-APC, Human (Miltenyi Biotec, clone: ​​REA897, model number: 130-114-861) was used as the antibody against the CD90 antigen, and CD105-APC, Human (Miltenyi The following antibodies were used: CD166-APC, Human (Miltenyi Biotec, clone: ​​REA442, model number: 130-106-576) as the antibody against the CD166 antigen; CD45-APC, Human (Miltenyi Biotec, clone: ​​REA747, model number: 130-110-633) as the antibody against the CD45 antigen; and CD326-APC, Human (Miltenyi Biotec, clone: ​​REA764, model number: 130-111-000) as the antibody against the CD326 antigen. Surface antigen analysis was performed using Merck's Guava easyCyte, with the following measurement conditions: number of cells analyzed: 30,000 cells, flow rate setting: 35.4 μL / min. Furthermore, the ratio of positive cells to each antigen (positivity rate) was calculated using the following procedure.

[0108] (1) The measurement results of the isotype control were plotted as a dot plot with SSC on the vertical axis and FSC on the horizontal axis. (2) A gate was set for the cell population corresponding to mesenchymal cells, and a histogram was developed for that cell population with the number of cells on the vertical axis and the trend intensity of APC on the horizontal axis. (3) In the histogram of (2), all regions (gates) were selected in which the cell population with stronger fluorescence intensity accounted for 0.5% or less of the total cells measured with the isotype control antibody. (4) Of the total cells measured with antibodies corresponding to surface antigen markers, the proportion of cells contained within the gate selected in (2) was calculated.

[0109] (Steps 1-5: Long-term subculturing of mesenchymal cells) The cell population containing mesenchymal cells obtained in "Step 1-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" described above was placed in an adherent cell culture dish 90 (Sumitomo Bakelite Co., Ltd.) at a rate of 9,000 cells / cm². 2 Cells were seeded at the following density. After seeding, the cells were cultured in αMEM containing 10% FBS until subconfluent. After culturing, 2 mL of TrypLE Select was added to each 90°C adherent cell culture dish, and the cells were incubated at 37°C for 10 hours to completely detach the cell population. Subsequently, the cell population was divided into 500-9,000 cells / cm². 2 The cells were subcultured at a specific density until cell proliferation stopped. The proliferation curve is shown in Figure 1. As a result, mesenchymal stem cells were able to divide up to approximately 42 times.

[0110] <Example 2: hPL culture of amniotic mesenchymal cells I> (Step 2-1: Amniotic membrane harvesting) The amniotic membrane was obtained from the same donor (Donor #1) as in Example 1, using the same method as in Example 1.

[0111] (Step 2-2: Enzymatic treatment of amniotic membrane and collection of mesenchymal cells) A cell population containing mesenchymal cells was obtained using the same method as in Example 1.

[0112] (Steps 2-3: Culture of mesenchymal cells) The cell population containing mesenchymal cells obtained in "Step 2-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" described above was seeded in a CellSTACK® (Corning) culture vessel. The seeding density was 6,000 cells / cm³. 2The density was set to [density]. After cell seeding, the cells were adherently cultured in αMEM containing human platelet lysate (hPL) at a final concentration of 5% until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was the 0th passage. Subsequently, 1 / 5 of the cell population was seeded into CellSTACK® on the same scale as the previous culture, and subcultured in αMEM containing hPL at a final concentration of 5%. The culture medium was changed every 2-4 days. When subconfluent was reached, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was the 1st passage.

[0113] Subsequently, the cell concentration increased to 2 × 10 7 RPMI1640 was added to achieve a cell / mL ratio. An equal volume of CP-1(registered trademark) solution (CP-1(registered trademark): 25% human serum albumin = 34:16 ratio) was added to this solution, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 15,000-18,000 cells / cm³. 2 At the specified density, the cell population from the first passage was seeded onto CellSTACK® and adherently cultured in αMEM containing 5% human platelet lysate (hPL) until subconfluent. Subsequently, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was the second passage.

[0114] Next, 1 / 5 of the aforementioned cell population was seeded into CellSTACK® on the same scale as the previous culture, and subcultured in αMEM containing a final concentration of 5% hPL. The culture medium was changed every 2 to 4 days. When subconfluence was reached, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here was the 3rd passage.

[0115] Subsequently, the cell concentration was 4 × 10 6 RPMI1640 was added to achieve a cell / mL ratio. An equal volume of CP-1 solution (registered trademark) (a solution mixed with CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34:16) was added, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 6,000 cells / cm³. 2 At this density, the cell population of the third passage was seeded onto CellSTACK® and adherently cultured in αMEM containing 5% human platelet lysate (hPL) until subconfluent. Subsequently, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was a cell population of the fourth passage.

[0116] Next, 1 / 5 of the cell population was seeded into CellSTACK® on the same scale as the previous culture, and subcultured in αMEM containing 5% hPL. The culture medium was changed every 2-4 days. When subconfluence was reached, 15 mL of TrypLE Select was added per CellSTACK® stack, and incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here was the 5th subgeneration. For the cell population, the cell concentration was 4 × 10⁶. 6RPMI1640 was added to achieve a cell / mL concentration. An equal volume of CP-1(registered trademark) solution (CP-1(registered trademark): 25% human serum albumin = 34:16 ratio) was added, and the samples were transferred to cryovials in 1 mL increments. These were then slowly frozen to -80°C and subsequently stored under liquid nitrogen.

[0117] (Step 2-4: Surface antigen analysis of mesenchymal cells) For a population of 5th passage mesenchymal cells cultured using the above culture method, various surface antigens (positive rates for CD324, CD73 (known as an MSC marker), CD90, CD105, CD166, CD45, and CD326) were analyzed using a flow cytometer. The results showed that the positive rates for CD324 and CD105 were over 70% (specifically CD324: 91%, CD105: 95%), and the positive rates for CD73, CD90, and CD166 were all over 90% (specifically CD73: 100%, CD90: 100%, CD166: 99%). The negative rates for CD45 and CD326 were all over 95% (specifically CD45: 100%, CD326: 99%). From these results, it was found that the cells cultured using the above method constitute a cell population including mesenchymal cells. Furthermore, it was confirmed that the cell population described in Example 1 was a cell population in which the proportion of mesenchymal cells exhibiting CD324 positivity was 70% or more, and the proportion of mesenchymal cells exhibiting CD90 positivity was 90% or more. The measurement method and reagents used are the same as in Example 1.

[0118] (Steps 2-5: Long-term subculturing of mesenchymal cells) The cell population containing mesenchymal cells obtained in "Step 2-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" described above was loaded into a CellSTACK® (Corning) at a rate of 1,000 cells / cm². 2Cells were seeded at the following density. After seeding, the cells were cultured in αMEM containing 5% hPL until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK® stack, and the cells were incubated at 37°C for 10 hours to completely detach the cell population. Subsequently, the cells were divided into 1,000 cells / cm². 2 The cells were subcultured at a specific density until cell proliferation stopped. The proliferation curve is shown in Figure 2. As a result, mesenchymal stem cells were able to divide up to approximately 84 times.

[0119] <Example 3: hPL culture of amniotic mesenchymal cells II> In Example 3, described below, a mesenchymal cell population was obtained with different donor, enzyme treatment conditions, and culture conditions compared to Examples 1 and 2. The amniotic membrane and placenta, which are fetal appendages, were aseptically collected from three pregnant women (donors #2 to #4) who underwent elective cesarean section and had given informed consent, unlike in Examples 1 and 2.

[0120] (Step 3-1: Amniotic membrane harvesting) The amniotic membrane was obtained using the same method as in Example 1 and Example 2.

[0121] (Step 3-2: Enzymatic treatment of amniotic membrane and collection of mesenchymal cells) The amnion, containing both epithelial and mesenchymal cell layers, was enzymatically treated by immersing it in a Hanks equilibrium salt solution (containing Ca·Mg) with 480 PU / mL collagenase and 400 PU / mL dispase I, and shaking it at 50 rpm at 37°C for 90 minutes. After enzymatic treatment, the solution was filtered through a 95 μm nylon mesh to remove undigested amnional material, and a cell suspension containing mesenchymal cells was recovered.

[0122] (Step 3-3: Culture of mesenchymal cells) The cell population containing mesenchymal cells obtained in "Step 3-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" described above was seeded into the CellSTACK® culture vessel. The seeding density was 1,000 cells / cm². 2The following steps were taken. After cell seeding, the cells were cultured in αMEM containing 5% human platelet lysate (hPL) until subconfluent. The culture medium was changed every 3-5 days. After culturing, 15 mL of TrypLE Select was added to each CellStack® stack and incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was passage 0. Subsequently, the cell concentration was increased to 2 × 10⁶. 7 Physiological saline was added to achieve a cell / mL ratio. An equal volume of CP-1® solution (CP-1®: 25% human serum albumin = 34:16 ratio) was added, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 1,000 cells / cm³. 2 First-passage cell populations were seeded on CellSTACK® at a density and adherently cultured for 5 days in αMEM containing a final concentration of 5% human platelet lysate (hPL) until subconfluent.

[0123] Subsequently, 15 mL of TrypLE Select was added to each CellStack® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here is the first passage. Next, the cell concentration was 2 × 10⁻⁶. 7 Physiological saline was added to achieve a cell / mL ratio. An equal volume of CP-1® solution (CP-1®: 25% human serum albumin = 34:16 ratio) was added, and 1 mL portions were transferred to cryovials. These were then slowly frozen to -80°C and stored under liquid nitrogen for 1 day. Subsequently, the frozen cell population was thawed, yielding approximately 1,000 cells / cm³. 2 Cell populations from the second passage were seeded at a density onto CellSTACK® and cultured in αMEM containing 5% human platelet lysate (hPL) for 5 days until subconfluent.

[0124] Subsequently, 15 mL of TrypLE Select was added to each CellStack® stack, and the cells were incubated at 37°C for 3 minutes, allowing for complete detachment of the cell population. The cell population obtained here was a 2nd passage cell population. For the cell population, the cell concentration was 4 × 10⁶. 6 Physiological saline was added to achieve a concentration of cells / mL. An equal volume of CP-1 solution (registered trademark) (a solution mixed with CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34:16) was added, and the samples were transferred to cryovials in 1 mL portions. These were slowly frozen to -80°C and then stored under liquid nitrogen.

[0125] (Steps 3-4: Surface antigen analysis of mesenchymal cells) For cell populations of the second passage (#2~#4) cultured using the culture method described in Step 3-3, various surface antigens (positive rates for CD324, CD73, CD90, CD105, CD166, CD45, and CD326) were analyzed using a flow cytometer. As a result, the positive rates for CD324 and CD105 were 70% or higher (specifically, for #2, #3, and #4, in that order: CD324: 90%, 87%, 87%; CD105: 89%, 91%, 74%), and the positive rates for CD73, CD90, and CD166 were all 90% or higher (specifically, for #2, #3, and #4, in that order: CD73: 100%, 99%, 100%; CD90: 100%, 99%, 100%; CD166: 99%, 97%, 98%). The negative rates for both CD45 and CD326 were 95% or higher (specifically, for #2, #3, and #4, the rates were CD45: 99%, 100%, 100%, and CD326: 99%, 100%, 100%). From these results, it was found that the cell populations #2, #3, and #4 cultured using the above culture method all contained mesenchymal cells that were positive for CD324. Furthermore, it was confirmed that the cell population described in Example 3 was a cell population in which the proportion of mesenchymal cells that were positive for CD324 was 70% or higher, and the proportion of mesenchymal cells that were positive for CD90 was 90% or higher. The measurement method and reagents used were the same as in Example 1.

[0126] <Example 4: Evaluation of the efficacy of amniotic mesenchymal cells in mdx mice (DMD model mice)> (Step 4-1: Administration of amniotic mesenchymal cells to MDX mice) The cell population (#3) of cryopreserved 2nd passage amniotic mesenchymal cells (including amniotic mesenchymal stem cells (hAMSCs)) obtained in "Step 3-3: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" of Example 3 was thawed, yielding approximately 6,000 cells / cm². 2 At a density of 225cm 2 Cells were seeded in tissue culture flasks and cultured in αMEM containing 5% human platelet lysate (hPL) for 5 days until subconfluent. Then, 2.5 mL of 0.25% trypsin (containing EDTA) was added per flask, and the mixture was incubated at room temperature for 1 minute to completely detach the cell population. A homogeneous cell suspension was obtained using a cell filtration filter (60 μm cell strainer), and the cells were resuspended in PBS. (8 × 10⁶ cells) 5 Prepared in 100 μL of cells.

[0127] The cell suspension prepared above was administered via tail vein to mdx / C57BL / B10 mice (4-5 weeks old males, body weight ≥10g, obtained from CREA Japan Co., Ltd.) once a week for 4 or 6 weeks.

[0128] (Step 4-2: Grip strength evaluation) The forelimb grip strength of MDX mice (1 year old) administered amniotic mesenchymal stem cells was measured using the Saito-type mouse grip strength measurement device. In this study, wild-type mice and untreated MDX mice (both 1 year old) were also measured as controls. The results are shown in Figure 3. MDX mice administered amniotic mesenchymal stem cells four or six times showed improved grip strength reduction compared to normal MDX mice.

[0129] (Process 4-3: Circulatory function evaluation) Left ventricular function was evaluated in mdx mice (1 year old) administered amniotic mesenchymal stem cells using an ultrasound imaging system (GE Healthcare, VividS60RN, 12Hz probe). In this evaluation, left ventricular volume was estimated by measuring the diastolic and systolic diameters of the left ventricle under 2% isoflurane inhalation anesthesia, and the left ventricular diameter shortening ratio (LVFS (%)) was calculated. In this study, measurements were also taken in wild-type mice and untreated mdx mice (both 1 year old) as controls. The results are shown in Figure 4. In mdx mice administered amniotic mesenchymal stem cells four times, LVFS was significantly improved compared to normal mdx mice. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A muscular dystrophy treatment agent containing a cell population, including mesenchymal cells derived from amniotic tissue, as an active ingredient.

2. The aforementioned cell population, The muscular dystrophy treatment agent according to claim 1, wherein the proportion of CD324-positive mesenchymal cells in the cell population is 70% or more, and the proportion of CD90-positive mesenchymal cells is 90% or more.

3. The muscular dystrophy treatment agent according to claim 1 or 2, wherein the proportion of cells exhibiting CD326 positivity in the aforementioned cell population is 10% or less.

4. A muscular dystrophy treatment agent according to any one of claims 1 to 3, wherein in the cell population, the proportion of mesenchymal cells exhibiting CD73 positivity is 80% or more, the proportion of mesenchymal cells exhibiting CD166 positivity is 80% or more, the proportion of cells exhibiting CD45 positivity is 10% or less, and the proportion of mesenchymal cells exhibiting CD105 positivity is 70% or more.

5. The muscular dystrophy treatment agent according to any one of claims 1 to 4, wherein the cell population is obtained from a living organism different from the subject to which the agent is administered.

6. The muscular dystrophy treatment agent according to any one of claims 1 to 4, wherein the cell population is obtained from a living organism of the same species as the subject to administration.

7. A muscular dystrophy treatment agent according to any one of claims 1 to 6, wherein the target recipient is a human.

8. The muscular dystrophy treatment agent according to any one of claims 1 to 7, wherein the aforementioned cell population is of human origin.

9. The muscular dystrophy treatment agent according to any one of claims 1 to 8, wherein the mesenchymal cells have no or low ability to differentiate into osteoblasts and adipocytes.

10. The muscular dystrophy treatment agent according to any one of claims 1 to 9, wherein the mesenchymal cells have an anti-inflammatory effect.

11. The muscular dystrophy treatment agent according to any one of claims 1 to 10, wherein the cell population is a population of cells cultured using a culture medium containing platelet lysate.

12. A muscular dystrophy treatment agent according to any one of claims 1 to 11, which is administered intravenously.

13. A muscular dystrophy treatment agent according to any one of claims 1 to 12, wherein the therapeutic effect after administration lasts for six months or more.

14. A muscular dystrophy treatment agent according to any one of claims 1 to 13, used for the treatment of Duchenne muscular dystrophy.

15. (a) A step of preparing a cell population containing mesenchymal cells from amniotic tissue; and (b) A step of culturing the cell population from (a), A method for manufacturing a muscular dystrophy treatment agent, including [the specified ingredient].

16. The method according to claim 15, wherein a culture medium containing platelet lysate is used for the culture in (b) above.

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