Method for producing proliferating cells, method for producing cell products, mesenchymal stem cell population and method for producing the same, stem cell culture supernatant and method for producing the same, and therapeutic agent

Abstract

To provide a cell culture technology that enables the proliferation of cells seeded at a low cell density at a high growth rate. [Solution] A method for producing proliferating cells, comprising growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, thereby increasing the cell population.

Description

[Technical Field]

[0001] The present invention relates to a method for producing proliferating cells, a method for producing a cell product, a mesenchymal stem cell population and a method for producing the same, a stem cell culture supernatant and a method for producing the same, and a therapeutic agent. [Background Art]

[0002] Stem cells such as mesenchymal stem cells have attracted worldwide attention for their application in regenerative medicine. For the application of stem cells to regenerative medicine, it is necessary to develop a culture technique that can stably culture and proliferate these cells.

[0003] In order to culture, proliferate, and mass produce stem cells, the common practice is: first seed the cells in a culture vessel at a constant cell density, culture until 80-90% or more of the surface area of the culture vessel is covered with cells (a so-called confluent state), then detach the cells with an enzyme such as trypsin, and passage the obtained cells to a plurality of new culture vessels (see, for example, Patent Document 1).

[0004] On the other hand, stem cells such as mesenchymal stem cells are known to secrete cell-derived components such as various cytokines and exosomes, and are expected for application in medicine. [Prior Art Literature] [Patent Literature]

[0005] [Patent Document 1] International Publication No. 2014 / 035215 [Summary of the Invention] [Problem to be Solved by the Invention]

[0006] When mass-producing proliferating cells or cell-derived components using cell culture technology, it is required to efficiently proliferate cells and efficiently recover the culture supernatant containing cell-derived components.

[0007] Therefore, the present invention aims to provide a cell culture technology that can propagate cells seeded at a low cell density at a high proliferation rate. Furthermore, the present invention aims to provide a cell culture technology that further develops the above-mentioned cell culture technology, and that can produce a large amount of cell products by a simple method while maintaining the adhesion state of the obtained proliferating cells. Furthermore, the present invention aims to provide a mesenchymal stem cell population having novel characteristics based on the cell culture technology described above, and to provide a stem cell culture supernatant containing a large amount of cell products such as cytokines, based on the cell culture technology described above. The present invention also aims to provide a therapeutic agent containing the above-described mesenchymal stem cell population or the above-described culture supernatant. [Means for solving the problem]

[0008] In one aspect, a method for producing proliferating cells is provided, which includes growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture.

[0009] From another perspective, The process involves culturing cells in a growth culture medium according to the "Method for Producing Proliferating Cells" described above, to obtain proliferating cells in an adherent state, and The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. A method for producing cell products, including the above, is provided. From yet another perspective, The process involves culturing cells in a growth culture medium according to the "Method for Producing Proliferating Cells" described above, to obtain proliferating cells in an adherent state, and The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method for producing cell products is provided, wherein the cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells.

[0010] In another aspect, a method is provided for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a proliferation culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants to obtain proliferating cells. From another perspective, a mesenchymal stem cell population is provided in which the proportion of HLA-ABC-positive mesenchymal stem cells is 70% or less. From yet another perspective, A method for producing the culture supernatant of stem cells, Stem cells are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method is provided in which the culturing in the production culture medium and the culturing in the recovery culture medium are alternately repeated while maintaining the adhesion state of the cells, and the method further includes collecting the supernatant of the production culture medium after the culturing in the production culture medium. From another perspective, stem cell culture supernatant containing 5000 pg / mL or more of HGF is provided. From another perspective, stem cell culture supernatant containing CD9 / CD63 EC domain fusion protein at a concentration of 50 pg / mL or higher is provided. From another perspective, therapeutic agents comprising the aforementioned mesenchymal stem cell population or the aforementioned culture supernatant are provided. [Effects of the Invention]

[0011] According to the present invention, a cell culture technique capable of proliferating cells seeded at a low cell density at a high proliferation rate can be provided. Further, according to the present invention, which is a cell culture technique obtained by further developing the above cell culture technique, a cell culture technique capable of producing a large amount of cell products by a simple method while maintaining the adherent state of the obtained proliferated cells can be provided. According to the present invention, based on the above-mentioned cell culture technique, a mesenchymal stem cell population having novel characteristics can be provided, and a stem cell culture supernatant containing a large amount of cell products such as cytokines can be provided. Further, according to the present invention, a therapeutic agent comprising the above-mentioned mesenchymal stem cell population or the above-mentioned culture supernatant can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Figure 1] Graph showing the number of proliferated cells (Comparative Example). [Figure 2] Micrograph of cells after 20 days of culture at a seeding density of 10 cells / cm 2 (Comparative Example). [Figure 3] Graph showing the number of proliferated cells (Example of the present invention). [Figure 4] Micrograph of cells after 20 days of culture at a seeding density of 10 cells / cm 2 (Example of the present invention). [Figure 5] Graph showing the number of proliferated cells (Reference Example). [Figure 6] Micrograph of cells after 20 days of culture at a seeding density of 10 cells / cm 2 (Reference Example). [Figure 7A] Micrograph of cells after 3 days of production culture (Comparative Example). [Figure 7B] Micrograph of cells after 3 days of production culture (Comparative Example). [Figure 8A] Micrograph of cells after 3 days of production culture (Example of the present invention). [Figure 8B] Micrograph of cells after 3 days of production culture (Example of the present invention). [Figure 9]A schematic diagram showing the culture process performed in Example 3. [Figure 10] A graph showing the results of cytokine quantification. [Figure 11] A graph showing the results of cytokine quantification. [Figure 12] A graph showing the results of cytokine quantification. [Figure 13] A graph showing the positivity rate of cell surface markers in umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragments. [Figure 14] A graph showing the positivity rate of cell surface markers in adipose-derived mesenchymal stem cells cultured in the presence of laminin fragments. [Figure 15] A graph showing the positivity rate of cell surface markers in umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragments. [Figure 16] A graph showing the amount of HGF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 17] A graph showing the amount of HGF contained in the culture supernatant of adipose-derived mesenchymal stem cells. [Figure 18] A graph showing the amount of MCP-1 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 19] A graph showing the amount of GRO / CXCL1 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 20] A graph showing the amount of fibronectin contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 21] A graph showing the amount of PDGF-AA contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 22] A graph showing the amount of VEGF contained in the culture supernatant of adipose-derived mesenchymal stem cells. [Figure 23] A graph showing the amount of TGF-1β contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 24] A graph showing the amount of IL-4 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 25] A graph showing the amount of IL-10 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 26] A graph showing the amount of IL-13 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 27] A graph showing the amount of IL-7 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 28] A graph showing the amount of IL-15 contained in the culture supernatant of adipose-derived mesenchymal stem cells. [Figure 29] A graph showing the amount of IL-9 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 30] A graph showing the amount of IL-1α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 31] A graph showing the amount of IL-1β contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 32] A graph showing the amount of TNF-α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 33] A graph showing the amount of IL-8 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 34] A graph showing the amount of EOTAXIN contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 35] A graph showing the amount of IL-6 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 36] A graph showing the amount of G-CSF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 37] A graph showing the amount of GM-CSF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 38] A graph showing the amount of MCP-3 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 39] A graph showing the amount of IL-12P40 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 40] A graph showing the amount of IP-10 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 41] A graph showing the amount of MIP-1α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 42] A graph showing the amount of exosome marker proteins in the culture supernatant of umbilical cord-derived mesenchymal stem cells. [Figure 43] A graph showing the ratio of blood flow in the lower extremities. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below, but the following description is intended to explain the present invention in detail and is not intended to limit it.

[0014] When cells are propagated by subculturing, the lower the cell density at seeding, the more culture vessels will be used for the next generation from the cells in one culture vessel. If the so-called split ratio is 1:10, cells that have reached confluence in one culture vessel can be seeded into 10 culture vessels, and if the split ratio is 1:100, cells that have reached confluence in one culture vessel can be seeded into 100 culture vessels. However, the typical split ratio for stem cells is around 1:5 to 1:20, and stem cells are rarely seeded at low cell densities to achieve split ratios of 1:100 or 1:1000. In reality, stem cells are seeded and subculturised at cell densities exceeding 2000 cells / cm2.

[0015] On the other hand, research is being conducted on culturing human mesenchymal stem cells to produce cell-derived components (hereinafter also referred to as cell products) such as various cytokines and exosomes, and administering these to humans for disease treatment. In this case, it is undesirable to culture stem cells in a culture medium containing animal-derived components, human serum, or recombinant proteins such as foreign cytokines and insulin, and then purify and use the resulting supernatant. This is because there is a concern that the above-mentioned foreign components, other than cell-derived components, may enter the human body. Therefore, it is desirable to use cell culture supernatant obtained by culturing stem cells in a protein-free culture medium that does not contain foreign components for treatment.

[0016] Under these technical circumstances, the inventors found that when stem cells were seeded and cultured at a cell density of 10,000 cells / cm2, they were able to proliferate the cells to a confluent state. However, when stem cells were seeded and cultured at a lower cell density of 1,000 cells / cm2 or less, cell proliferation stopped, and they could not be proliferated to a confluent state (see Example 1, Figures 1 and 2 described below). Furthermore, when the inventors seeded and cultured stem cells at a cell density of 1,000 cells / cm2 and cultured the resulting proliferating cells in a protein-free culture medium that did not contain any foreign components (i.e., performed production culture), the cells detached from the culture vessel during production culture, and the adhesion state of the proliferating cells could not be maintained (see Example 2, Figures 7A and 7B described below).

[0017] The inventors have worked to solve these problems. As a result, firstly, the inventors have found that even when stem cells are seeded at a low cell density, they can be proliferated to a confluent state when cultured in the presence of a laminin fragment having integrin-binding activity (hereinafter also simply referred to as laminin fragment) (see Example 1, Figures 3 and 4 below). Secondly, the inventors have found that when stem cells are cultured and proliferated in the presence of a laminin fragment, the resulting proliferated cells can be cultured in a protein-free culture medium that does not contain foreign components, and the cells do not detach from the culture vessel during the culture process, maintaining the adhesion state of the proliferated cells (see Example 2, Figures 8A and 8B below). Thirdly, the inventors found that when the above-mentioned proliferating cells were cultured in a protein-free culture medium that did not contain foreign components (i.e., production culture was performed), and then further cultured in a cell proliferation culture medium (i.e., recovery culture was performed), and production culture was performed again, it was possible to repeatedly perform production culture while maintaining the adhesion state of the proliferating cells, and to produce cell-derived components over a long period of time (see Example 3 below).

[0018] Based on these discoveries, the present inventors have completed the present invention. The methods of the present invention, namely "method for producing proliferating cells" and "method for producing cell products," will be described below in this order.

[0019] <1. Method for producing proliferating cells> In one aspect, a method for producing proliferating cells is provided, which includes growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture.

[0020] Preferably, the culture can be carried out in a growth culture medium containing the culture substrate (i.e., a laminin fragment having integrin-binding activity or a variant thereof). In other words, according to a preferred embodiment, a method for producing proliferating cells is provided, which includes growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 by adherent culture in a growth culture medium containing a laminin fragment having integrin-binding activity (hereinafter also simply referred to as a laminin fragment) or a variant thereof, thereby increasing the cell population.

[0021] To give a specific example, the method for producing proliferating cells is: A growth culture medium containing laminin fragments or their variants is prepared in advance, cells are suspended in the prepared growth culture medium to a seeding density of 0.002 to 2000 cells / cm2, and the resulting cell suspension is seeded into a culture vessel. It is possible to culture the cells in the growth medium by adherent culture to increase their size.

[0022] Alternatively, to give another specific example, the method for producing proliferating cells is: The cells are suspended in a growth medium to a seeding density of 0.002 to 2000 cells / cm2, laminin fragment or a modified version thereof is added to the resulting cell suspension, and this is then seeded into a culture vessel. It is possible to culture the cells in the growth medium by adherent culture to increase their size.

[0023] Alternatively, to give another specific example, the method for producing proliferating cells is: The cells are suspended in a growth medium to a seeding density of 0.002 to 2000 cells / cm2, the resulting cell suspension is seeded in a culture vessel, and laminin fragments or their variants are added to the seeded cell suspension. It is possible to culture the cells in the growth medium by adherent culture to increase their size.

[0024] (cell) Any cells can be used as the cells, such as stem cells. Preferably, the cells are mesenchymal stem cells, induced pluripotent stem cells (iPS cells), or embryonic stem cells (ES cells), and more preferably mesenchymal stem cells. More preferably, the cells are umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, or umbilical cord blood-derived mesenchymal stem cells, and even more preferably umbilical cord-derived mesenchymal stem cells.

[0025] In a preferred embodiment, the cells are human cells; that is, the cells are, for example, human stem cells. Preferably, the cells are human mesenchymal stem cells, human induced pluripotent stem cells (iPS cells), or human embryonic stem cells (ES cells), and more preferably human mesenchymal stem cells. More preferably, the cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells, and even more preferably human umbilical cord-derived mesenchymal stem cells.

[0026] The cells may be frozen cells. That is, the cells may be cells prepared by thawing frozen cells. When it is desired to produce proliferating cells or cell-derived components, by using cryopreserved cells as starting cells and proliferating them according to the method of the present invention, proliferating cells or cell-derived components can be produced when needed.

[0027] (seeding) In this method, cells are seeded into the growth culture medium at a cell density of 0.002 to 2000 cells / cm2.

[0028] The number of cells is preferably 0.002 to 1900 cells / cm2, more preferably 0.002 to 1800 cells / cm2, even more preferably 0.002 to 1700 cells / cm2, even more preferably 0.002 to 1600 cells / cm2, even more preferably 0.002 to 1500 cells / cm2, even more preferably 0.002 to 1400 cells / cm2, even more preferably 0.002 to 1300 cells / cm2, even more preferably 0.002 to 1200 cells / cm2, even more preferably 0.002 to 1100 cells / cm2, and even more preferably 0.002 Seeding is performed at a cell density of ~1000 cells / cm2, more preferably 0.002~900 cells / cm2, more preferably 0.002~800 cells / cm2, more preferably 0.002~700 cells / cm2, more preferably 0.002~600 cells / cm2, more preferably 0.002~500 cells / cm2, more preferably 0.002~400 cells / cm2, more preferably 0.002~300 cells / cm2, more preferably 0.002~200 cells / cm2, and more preferably 0.002~100 cells / cm2.

[0029] Taking into account the time it takes for the cells to reach confluence, the cell density is preferably 1 to 2000 cells / cm2, more preferably 1 to 1900 cells / cm2, even more preferably 1 to 1800 cells / cm2, even more preferably 1 to 1700 cells / cm2, even more preferably 1 to 1600 cells / cm2, even more preferably 1 to 1500 cells / cm2, even more preferably 1 to 1400 cells / cm2, even more preferably 1 to 1300 cells / cm2, even more preferably 1 to 1200 cells / cm2, and even more preferably The seeds are seeded at a cell density of 1 to 1100 cells / cm2, more preferably 1 to 1000 cells / cm2, more preferably 1 to 900 cells / cm2, more preferably 1 to 800 cells / cm2, more preferably 1 to 700 cells / cm2, more preferably 1 to 600 cells / cm2, more preferably 1 to 500 cells / cm2, more preferably 1 to 400 cells / cm2, more preferably 1 to 300 cells / cm2, more preferably 1 to 200 cells / cm2, and more preferably 1 to 100 cells / cm2.

[0030] The cell densities mentioned above are lower than the seeding densities typically used when culturing stem cells. Cells are usually seeded as single cells. Single cells can be prepared by treating cell aggregates with proteolytic enzymes (e.g., trypsin).

[0031] Depending on the type of cell, a known cell proliferation culture medium can be used. For example, in the case of human stem cells, a commercially available culture medium for human stem cell proliferation can be used.

[0032] Considering that the cultured cells and culture medium will be used for therapeutic purposes such as disease treatment and regenerative medicine, it is desirable that the culture medium be serum-free and free of heterogeneous components (xeno-free).

[0033] Furthermore, while the "production culture medium" described later is preferably free of proteins, the growth culture medium is preferably free of proteins that promote cell proliferation. Examples of proteins that promote stem cell proliferation include bFGF (basic fibroblast growth factor), TGFβ1 (transforming growth factor β1), EGF (epidermal growth factor), IGF (insulin-like growth factor), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), insulin, albumin, and transferrin. More specifically, the growth culture medium may be a medium prepared by adding growth factors to a basic cell culture medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.).

[0034] For human mesenchymal stem cells, growth culture media such as MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Corporation), Mesenchymal Stem Cell Growth Medium DXF (Takara Bio Inc.), and MSC NutriStem XF Medium (Biological Industries) can be used. All of these growth culture media are serum-free and xeno-free (doseno-free) media.

[0035] Any container used for adherent cell culture can be used as the culture vessel. Generally, flat-bottomed containers such as culture flasks, culture dishes, and culture plates can be used. Using a culture vessel with a large base area allows for the production of a large quantity of proliferating cells or cell-derived components from a single culture vessel. For example, it is desirable to use a culture vessel with a base area of ​​500 cm² or more, preferably 500 to 10000 cm².

[0036] (Culturing) Cells seeded at a cell density of 0.002 to 2000 cells / cm2 are cultured in a growth medium by adherent culture in the presence of laminin fragments or their variants that have integrin-binding activity.

[0037] As described above, in the present invention, when stem cells are cultured in the presence of a "laminin fragment having integrin-binding activity (hereinafter also simply referred to as laminin fragment) or a modified version thereof," the stem cells can be proliferated to a confluent state even when seeded at a low cell density (see Example 1, Figures 3 and 4 below).

[0038] Therefore, it is desirable to continue culturing until the cells reach a confluent state. In this specification, “confluent state” means that the cells cover 80% or more of the bottom surface area of ​​the culture vessel. More preferably, culturing can be continued until the cells cover 80-90% of the bottom surface area of ​​the culture vessel.

[0039] Culturing in the presence of laminin fragments or their variants may be carried out using a growth culture medium containing laminin fragments or their variants, or using a culture vessel pre-coated with laminin fragments or their variants. Culturing using a growth culture medium containing laminin fragments or their variants is preferred over culturing using a culture vessel pre-coated with laminin fragments or their variants because it requires less laminin fragment or its variants and is simpler to perform.

[0040] Therefore, the culture can preferably be carried out in a growth culture medium containing a laminin fragment having integrin-binding activity or a variant thereof.

[0041] Laminin fragments known to possess integrin-binding activity can be used. It has been reported that when "laminin fragments with integrin-binding activity" are pre-coated onto culture vessels, human ES cells and human iPS cells can be cultured without the use of feeder cells (e.g., Nakagawa et al., Scientific Reports 4, Article Number: 3594 (2014) and International Publication No. 2011 / 043405). Culture without feeder cells is called feeder-free culture, and its use is becoming widespread because it does not use heterologous components derived from animals. Therefore, laminin fragments used in the art to proliferate human ES cells and human iPS cells in feeder-free culture can also be used in the method of the present invention.

[0042] Since it is preferable that the growth culture medium does not contain heterologous components, the laminin fragment is preferably derived from humans.

[0043] The laminin fragment is preferably a laminin E8 fragment, more preferably a laminin 511 E8 fragment. Laminin 511 E8 fragment is commercially available from Nippi Corporation under the trade name iMatrix-511 and can be suitably used. Laminin is composed of three subunit chains: an α chain, a β chain, and a γ chain. Five types of α chains (α1-α5), three types of β chains (β1-β3), and three types of γ chains (γ1-γ3) are known. Laminin 511 refers to laminin composed of α5, β1, and γ1.

[0044] Other laminin fragments that can be used include, for example, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment.

[0045] As modifiers of laminin fragments, known complexes consisting of a laminin fragment having integrin-binding activity and other functional molecules can be used, for example, a complex of a laminin fragment having integrin-binding activity and a cell adhesion molecule, or a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule (see WO2012 / 137970, WO2014 / 103534, and WO2016 / 010082).

[0046] Preferably, a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule can be used as a modified laminin fragment. The laminin fragment contained in such a complex can be the laminin fragment described above. The growth factor-binding molecule contained in such a complex is preferably heparan sulfate. Such a complex has growth factor-binding activity in addition to integrin-binding activity.

[0047] A modified laminin fragment is more preferably a complex of laminin E8 fragment and a growth factor binding molecule. The laminin E8 fragment contained in such complex is preferably the laminin E8 fragment exemplified above. The growth factor binding molecule contained in such complex is preferably heparan sulfate. Therefore, a modified laminin fragment is even more preferably a complex of laminin E8 fragment and heparan sulfate exemplified above. Most preferably, a modified laminin fragment is a complex of laminin 511 E8 fragment and heparan sulfate, or a complex of laminin 421 E8 fragment and heparan sulfate.

[0048] A complex of a laminin fragment with integrin-binding activity and a growth factor-binding molecule can be produced as a recombinant protein using known genetic recombination techniques.

[0049] For example, the concentration of laminin fragment or its variant in the growth culture medium can be 0.005 μg to 2 μg per 1 cm² of culture area in the culture vessel. Preferably, the concentration of laminin fragment or its variant in the growth culture medium can be 0.01 μg to 0.5 μg per 1 cm² of culture area in the culture vessel. More preferably, the concentration of laminin fragment or its variant in the growth culture medium can be 0.05 μg to 0.25 μg per 1 cm² of culture area in the culture vessel.

[0050] Here, if the volume of the growth culture medium is, for example, 200 μl / cm² (culture area), then 0.005 μg / cm² to 2 μg / cm² corresponds to 0.025 μg / ml to 10 μg / ml, 0.01 μg / cm² to 0.5 μg / cm² corresponds to 0.05 μg / ml to 2.5 μg / ml, and 0.05 μg / cm² to 0.25 μg / cm² corresponds to 0.25 μg / ml to 1.25 μg / ml.

[0051] As described above, cultivation can be carried out in any culture vessel, for example, in a culture vessel having a bottom area of ​​500 cm² or more, preferably 500 to 10,000 cm². During cultivation, the growth culture medium may be replaced with a new one of the same composition as appropriate.

[0052] (effect) Conventionally, when cells were seeded at a low cell density, cell proliferation stopped, and it was not possible to grow them to a confluent state. However, with the method described above, cells seeded at a low cell density can be grown at a high proliferation rate. This makes it possible to increase the number of culture vessels used for the next generation from the cells in one culture vessel, and to efficiently produce proliferating cells and cell-derived components.

[0053] <2. Method for producing cell products> <2-1. First Embodiment (Embodiment in which a single production culture process is performed)> From another perspective, this involves culturing cells in a growth culture medium according to the "method for producing proliferating cells" described above, and obtaining proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. A method for producing cell products, including the above, is provided.

[0054] In other words, the method of producing cellular products is Cells seeded at a cell density of 0.002 to 2000 cells / cm2 are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments and their variants that have integrin-binding activity, by adherent culture, thereby obtaining proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. Includes.

[0055] In the following explanation, cultivation in a growth culture medium will be referred to as "growth culture," and cultivation in a production culture medium will be referred to as "production culture."

[0056] As described above, preferably, the growth culture can be carried out in a growth culture medium containing a laminin fragment having integrin-binding activity or a modified version thereof. That is, according to a preferred embodiment, cells seeded at a cell density of 0.002 to 2000 cells / cm2 are cultured by adherent culture in a growth culture medium containing a laminin fragment having integrin-binding activity or a modified version thereof to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. A method for producing cell products, including the above, is provided.

[0057] (Culturing) The process up to proliferation culture can be carried out as described in section <1. Method for Producing Proliferating Cells>. This allows for obtaining proliferating cells in an adherent state. If the proliferation culture is carried out until the cells reach a confluence state, proliferating cells in a confluence state can be obtained.

[0058] (Production culture) After proliferation culture, the proliferating cells are cultured in the production culture medium while maintaining their adherence. "Culturing the proliferating cells in the production culture medium while maintaining their adherence" means that after proliferation culture, the cells that are adhered to the bottom of the culture vessel are not detached from the bottom of the culture vessel and the production culture is carried out while maintaining their adherence.

[0059] Production culture can be performed by replacing the growth culture medium in the culture vessel with the production culture medium after the growth culture. This allows the proliferating cells to be cultured in the production culture medium while maintaining their adherence.

[0060] During production culture, cells can produce and release cellular products into the production culture medium. Cellular products are any substances that cells release into the production culture medium, such as cellular metabolites like amino acids, lipids, and sugars; hormones; peptides; secreted proteins such as cytokines and extracellular matrix; or exosomes. For example, stem cells can produce and release various cytokines and exosomes into the production culture medium. Therefore, the method for producing cellular products may further include a step of collecting the supernatant of the production culture medium after production culture.

[0061] The supernatant of the production culture medium obtained in the recovery process is intended for therapeutic use, such as disease treatment and regenerative medicine. Therefore, it is desirable that the production culture medium be free of heterologous components. Heterologous components refer to components derived from animals other than humans when the cultured cells are human cells. Furthermore, it is desirable that the production culture medium be free of cytokines and insulin. It is also desirable that the production culture medium be free of proteins. And it is also desirable that the production culture medium be free of serum.

[0062] More preferably, the production culture medium is a cell culture medium that does not contain heterologous components, cytokines, or insulin. Even more preferably, the production culture medium is a cell culture medium that does not contain heterologous components, cytokines, or insulin, and does not contain proteins. Even more preferably, the production culture medium is a cell culture medium that does not contain heterologous components, cytokines, or insulin, does not contain proteins, and does not contain serum.

[0063] The production culture medium can be a cell culture medium suitable for the cell type that does not contain the above-mentioned components (i.e., heterologous components, cytokines, insulin, proteins, and human serum). More specifically, the production culture medium may be a basic cell culture medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.), or a basic cell culture medium to which cell nutrients have been added. Basic cell culture media are commercially available and generally contain amino acids, vitamins, inorganic salts, and carbon sources. The production culture medium does not need to contain laminin fragments or their variants.

[0064] In the case of human mesenchymal stem cells, the production culture medium can be, for example, DMEM / F12 medium to which amino acids have been added. Commercially available amino acid solutions for culture medium addition can be used as the added amino acids, such as MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) or MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.). This production culture medium does not contain any heterologous components, cytokines, insulin, proteins, or human serum.

[0065] The production culture period is not particularly limited, but can be carried out for, for example, 0.5 to 10 days, preferably 2 to 5 days.

[0066] (effect) Conventionally, cell proliferation would stop during the growth culture process, or cells would detach from the culture vessel during the production culture process, making it impossible to maintain cell adhesion. In contrast, the method described above allows cells seeded at a low cell density to be grown at a high proliferation rate, and then production culture can be carried out while maintaining the adhesion of the resulting proliferated cells. This makes it possible to produce large quantities of cell products using a simple method.

[0067] Specifically, the method described above allows cells seeded at a low cell density to grow at a high proliferation rate, thus enabling the production of a large amount of cell products from a small amount of raw material cells. Furthermore, because the method maintains cell adhesion, it is convenient as it allows for a transition from proliferation culture to production culture simply by changing the culture medium. In other words, the method does not require cell subculturing, thereby eliminating lot-to-lot variations and subculturing costs. Moreover, because the method maintains cell adhesion, it is convenient as it allows for the easy collection of the culture supernatant containing cell products.

[0068] <2-2. Second Embodiment (Embodiment in which multiple production and cultivation processes are performed)> From another perspective, The process involves culturing cells in a growth culture medium according to the "Method for Producing Proliferating Cells" described above, to obtain proliferating cells in an adherent state, and The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method for producing cell products is provided, wherein the cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells.

[0069] In other words, the method of producing cellular products is Cells seeded at a cell density of 0.002 to 2000 cells / cm2 are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments and their variants that have integrin-binding activity, by adherent culture, thereby obtaining proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, The cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells.

[0070] In the following explanation, cultivation in a growth medium will be referred to as "growth culture," cultivation in a production medium will be referred to as "production culture," and cultivation in a recovery medium will be referred to as "recovery culture."

[0071] As described above, preferably, the growth culture can be carried out in a growth culture medium containing a laminin fragment having integrin-binding activity or a modified version thereof. That is, according to a preferred embodiment, cells seeded at a cell density of 0.002 to 2000 cells / cm2 are cultured by adherent culture in a growth culture medium containing a laminin fragment having integrin-binding activity or a modified version thereof to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method for producing cell products is provided, wherein the cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells.

[0072] (Culturing) The process up to proliferation culture can be carried out as described in section <1. Method for Producing Proliferating Cells>. This allows for obtaining proliferating cells in an adherent state. If the proliferation culture is carried out until the cells reach a confluence state, proliferating cells in a confluence state can be obtained.

[0073] (Production culture) The production culture process can be carried out as described in section <2-1. First Embodiment (Embodiment for Performing a Single Production Culture Process)>. This allows the cells to produce cellular products.

[0074] (Recovery culture) After production culture, the proliferating cells are cultured in a recovery culture medium while maintaining their adherence. "Culturing the proliferating cells in a recovery culture medium while maintaining their adherence" means that after production culture, the cells that are adhered to the bottom of the culture vessel are not detached from the bottom of the culture vessel and are cultured in a recovery culture while maintaining their adherence.

[0075] Recovery culture can be performed after production culture by replacing the production culture medium in the culture vessel with recovery culture medium. This allows the proliferating cells to be cultured in the production culture medium while maintaining their adhesion, and then further cultured in the recovery culture medium while maintaining their adhesion.

[0076] During recovery culture, cells can be restored to a state where they can once again produce sufficient amounts of cellular products. In other words, recovery culture is a process performed to restore the ability of cells to produce cellular products in preparation for the next production culture step.

[0077] Recovery culture can be performed using the culture medium used for cell proliferation culture. In other words, the recovery culture medium can be any known cell proliferation culture medium, depending on the type of cell. For example, in the case of human stem cells, a commercially available culture medium for human stem cell proliferation can be used.

[0078] Considering that the cultured cells and culture medium will be used for therapeutic purposes such as disease treatment and regenerative medicine, it is desirable that the recovery culture medium be serum-free and xeno-free (dosage-free) in type of culture medium.

[0079] Furthermore, while the "production culture medium" described above preferably does not contain proteins, the recovery culture medium preferably contains proteins that promote cell proliferation. Examples of proteins that promote stem cell proliferation include bFGF (basic fibroblast growth factor), TGFβ1 (transforming growth factor β1), EGF (epidermal growth factor), IGF (insulin-like growth factor), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), insulin, albumin, and transferrin. More specifically, the recovery culture medium may be a medium prepared by adding growth factors to a basic cell culture medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.).

[0080] The recovery culture medium may be a culture medium with the same composition as the growth culture medium. However, the recovery culture medium does not need to contain laminin fragments or their variants.

[0081] For human mesenchymal stem cells, recovery culture media such as MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Corporation), Mesenchymal Stem Cell Growth Medium DXF (Takara Bio Inc.), and MSC NutriStem XF Medium (Biological Industries) can be used. All of these recovery culture media are serum-free and xeno-free (dosage-free) media.

[0082] As described above, production culture and recovery culture can be repeated alternately while maintaining cell adhesion. The cycle of production culture and recovery culture can be repeated without limitation as long as the cells can recover their ability to produce cellular products. The cycle of production culture and recovery culture can be repeated, for example, 2 to 10 times. This allows the cells to produce cellular products and release them into the culture medium each time production culture is performed. Therefore, the method for producing cellular products may further include a step of collecting the supernatant of the production culture medium after the production culture.

[0083] The recovery culture period is not particularly limited, but can be carried out for, for example, 0.5 to 10 days, preferably 2 to 5 days.

[0084] (effect) The method according to the second embodiment, like the method according to the first embodiment, allows for the proliferation of cells seeded at a low cell density at a high proliferation rate, and then production culture can be performed while maintaining the adhesion state of the resulting proliferated cells. In addition, the method according to the second embodiment allows for recovery culture while maintaining the adhesion state of cells after production culture, thereby restoring the ability of the cells to produce cell products. This makes it possible to repeat the production culture process multiple times while maintaining the adhesion state of the cells, and as a result, large quantities of cell products can be produced over a long period of time using a simple method.

[0085] Specifically, the method according to the second embodiment allows cells seeded at a low cell density to be grown at a high proliferation rate, thus enabling the production of a large amount of cell products from a small amount of raw material cells. Furthermore, the method according to the second embodiment is convenient because it can maintain the cell adhesion state, allowing for a transition from proliferation culture to production culture, or the repetition of production culture and recovery culture cycles, simply by changing the culture medium. In other words, the method according to the second embodiment does not require cell subculturing, thereby eliminating lot-to-lot variations and costs associated with subculturing. Moreover, the method according to the second embodiment is convenient because it can maintain the cell adhesion state, allowing for easy recovery of the culture supernatant containing cell products.

[0086] In particular, the method according to the second embodiment has the advantage of enabling long-term substance production because it can produce cell products each time the production culture and recovery culture cycle is repeated. Furthermore, the method according to the second embodiment has the advantage of being able to continuously produce large quantities of cell products because the amount of cell products produced does not decrease even when the production culture and recovery culture cycle is repeated.

[0087] <3. Preferred Embodiments> Preferred embodiments of the present invention are summarized below. [A1] A method for producing proliferating cells, comprising growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture. [A2] A method for producing proliferating cells, comprising growing cells seeded at a cell density of 0.002 to 2000 cells / cm2 in a growth medium containing a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, thereby increasing the size of the cells. [A3] Seeding a cell suspension in a culture vessel containing a growth medium, a quantity of cells to achieve a seeding density of 0.002 to 2000 cells / cm2, and a culture substrate selected from laminin fragments having integrin-binding activity and their variants, and then, The cells are cultured in the growth medium by adherent culture to increase their size. A method for producing proliferating cells, including the production of proliferating cells. [A4] Seeding a cell suspension containing a growth medium and an amount of cells to achieve a seeding density of 0.002 to 2000 cells / cm2 into a culture vessel, To the seeded cell suspension, a culture substrate selected from laminin fragments having integrin-binding activity and their variants is added, and then, The cells are cultured in the growth medium by adherent culture to increase their size. A method for producing proliferating cells, including the production of proliferating cells. [A5] The method according to any one of [A1] to [A4], wherein the cell is a stem cell.

[0088] [A6] The method according to any one of [A1] to [A5], wherein the cells are mesenchymal stem cells, induced pluripotent stem cells (iPS cells), or embryonic stem cells (ES cells). [A7] The method according to any one of [A1] to [A6], wherein the cells are mesenchymal stem cells. [A8] The method according to any one of [A1] to [A7], wherein the cells are umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, or umbilical cord blood-derived mesenchymal stem cells. [A9] The method according to any one of [A1] to [A8], wherein the cells are umbilical cord-derived mesenchymal stem cells. [A10] The method according to any one of [A1] to [A5], wherein the cells are human stem cells.

[0089] [A11] The method according to any one of [A1] to [A6] and [A10], wherein the cells are human mesenchymal stem cells, human induced pluripotent stem cells (iPS cells), or human embryonic stem cells (ES cells). [A12] The method according to any one of [A1] to [A7], [A10] and [A11], wherein the cells are human mesenchymal stem cells. [A13] The method according to any one of [A1] to [A8] and [A10] to [A12], wherein the cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells. [A14] The method according to any one of [A1] to [A13], wherein the cells are human umbilical cord-derived mesenchymal stem cells. [A15] The method according to any one of [A1] to [A14], wherein the cells are frozen cells.

[0090] [A16] The method according to any one of [A1] to [A15], wherein the cells are cells prepared by thawing frozen cells. [A17] The number of cells is 0.002 to 1900 cells / cm2, preferably 0.002 to 1800 cells / cm2, more preferably 0.002 to 1700 cells / cm2, even more preferably 0.002 to 1600 cells / cm2, even more preferably 0.002 to 1500 cells / cm2, even more preferably 0.002 to 1400 cells / cm2, even more preferably 0.002 to 1300 cells / cm2, even more preferably 0.002 to 1200 cells / cm2, even more preferably 0.002 to 1100 cells / cm2, even more preferably 0.002 to 1000 cells / cm2, and The method according to any one of [A1] to [A16], wherein the cells are seeded at a cell density of preferably 0.002 to 900 cells / cm2, more preferably 0.002 to 800 cells / cm2, more preferably 0.002 to 700 cells / cm2, more preferably 0.002 to 600 cells / cm2, more preferably 0.002 to 500 cells / cm2, more preferably 0.002 to 400 cells / cm2, more preferably 0.002 to 300 cells / cm2, more preferably 0.002 to 200 cells / cm2, and more preferably 0.002 to 100 cells / cm2. [A18] The number of cells is 1 to 2000 cells / cm2, preferably 1 to 1900 cells / cm2, more preferably 1 to 1800 cells / cm2, even more preferably 1 to 1700 cells / cm2, even more preferably 1 to 1600 cells / cm2, even more preferably 1 to 1500 cells / cm2, even more preferably 1 to 1400 cells / cm2, even more preferably 1 to 1300 cells / cm2, even more preferably 1 to 1200 cells / cm2, even more preferably 1 to 1100 cells / cm2, even more preferably 1 to 1000 The method according to any one of [A1] to [A16], wherein the cells are seeded at a cell density of cells / cm2, more preferably 1 to 900 cells / cm2, more preferably 1 to 800 cells / cm2, more preferably 1 to 700 cells / cm2, more preferably 1 to 600 cells / cm2, more preferably 1 to 500 cells / cm2, more preferably 1 to 400 cells / cm2, more preferably 1 to 300 cells / cm2, more preferably 1 to 200 cells / cm2, and more preferably 1 to 100 cells / cm2. [A19] The method according to any one of [A1] to [A18], wherein the growth culture medium is a growth culture medium containing a protein that promotes the proliferation of the cells. [A20] The method according to any one of [A1] to [A19], wherein the growth culture medium is a culture medium containing a basic cell culture medium to which growth factors have been added.

[0091] [A21] The method according to any one of [A1] to [A20], wherein the growth medium is a basic cell culture medium to which growth factors have been added. [A22] The method according to any one of [A1] to [A21], wherein the culture is carried out until the cells reach a confluent state. [A23] The method according to any one of [A1] to [A22], wherein the culture is performed in a culture vessel having a bottom area of ​​500 cm2 or more. [A24] The method according to any one of [A1] to [A23], wherein the culture is performed in a culture vessel having a bottom area of ​​500 to 10000 cm2. [A25] The method according to any one of [A1] to [A24], wherein the culture substrate is a laminin fragment having integrin binding activity.

[0092] [A26] The method according to [A25], wherein the laminin fragment is a human-derived laminin fragment. [A27] The method according to [A25] or [A26], wherein the laminin fragment is a laminin E8 fragment. [A28] The method according to any one of [A25] to [A27], wherein the laminin fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [A29] The method according to any one of [A25] to [A28], wherein the laminin fragment is a laminin 511 E8 fragment. [A30] The method according to any one of [A1] to [A24], wherein the culture substrate is a modified laminin fragment having integrin-binding activity.

[0093] [A31] The method according to [A30], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and another functional molecule. [A32] The method according to [A30] or [A31], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule. [A33] The method according to [A31] or [A32], wherein the laminin fragment is a laminin E8 fragment. [A34] The method according to [A33], wherein the laminin E8 fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [A35] The method according to [A33] or [A34], wherein the laminin E8 fragment is a laminin 511 E8 fragment. [A36] The method according to [A33] or [A34], wherein the laminin E8 fragment is a laminin 421 E8 fragment.

[0094] [A37] The method according to any one of [A32] to [A36], wherein the growth factor binding molecule is heparan sulfate. [A38] The method according to any one of [A30] to [A37], wherein the modified product is a complex of laminin 511 E8 fragment and heparan sulfate. [A39] The method according to any one of [A30] to [A37], wherein the modified product is a complex of laminin 421 E8 fragment and heparan sulfate. [A40] The method according to any one of [A1] to [A39], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.005 μg to 2 μg per 1 cm² of culture area of ​​the culture vessel. [A41] The method according to any one of [A1] to [A40], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.01 μg to 0.5 μg per 1 cm² of culture area of ​​the culture vessel. [A42] The method according to any one of [A1] to [A41], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.05 μg to 0.25 μg per 1 cm² of culture area of ​​the culture vessel.

[0095] [B1] [A1]~[A42] The cells are cultured in a growth medium according to the method described in any one of the above, and the proliferating cells are obtained in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. A method for producing cellular products, including those mentioned above. [B2] The cells are cultured in a growth medium according to the method described in any one of [A1] to [A42] to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method for producing cell products, wherein the cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells. [B3] The method according to [B1] or [B2], wherein the cell product is a cell metabolite such as amino acids, lipids, sugars; hormones; peptides; secreted proteins such as cytokines, extracellular matrix; or exosomes. [B4] The method according to any one of [B1] to [B3], wherein the cell product is a cytokine. [B5] The method according to any one of [B1] to [B3], wherein the cell product is an exosome.

[0096] [B6] The method according to any one of [B1] to [B5], wherein the production culture medium is a culture medium that does not contain heterologous components. [B7] The method according to any one of [B1] to [B6], wherein the production culture medium is a culture medium that does not contain cytokines or insulin. [B8] The method according to any one of [B1] to [B7], wherein the production culture medium is a culture medium that does not contain protein. [B9] The method according to any one of [B1] to [B8], wherein the production culture medium is a serum-free culture medium. [B10] The method according to any one of [B1] to [B9], further comprising collecting the supernatant of the production culture medium after culturing in the production culture medium.

[0097] [B11] The method according to any one of [B1] to [B10], wherein the production culture medium is a culture medium containing a basic cell culture medium, or a culture medium containing a basic cell culture medium to which cell nutrients have been added. [B12] The method according to any one of [B1] to [B11], wherein the production culture medium is a basic cell culture medium or a basic cell culture medium to which cell nutrients have been added. [B13] The method according to any one of [B1] to [B12], wherein the production culture medium is a basic cell culture medium to which cell nutrients, preferably amino acids, have been added. [B14] The method according to any one of [B1] to [B13], wherein the production culture medium does not contain laminin fragments or their variants. [B15] The method according to any one of [B1] to [B14], wherein the culture in the production culture medium is carried out for 0.5 to 10 days, preferably 2 to 5 days.

[0098] [B16] The method according to any one of [B2] to [B15], wherein the recovery culture medium is the cell proliferation culture medium. [B17] The method according to any one of [B2] to [B16], wherein the recovery culture medium is a culture medium containing a protein that promotes the proliferation of the cells. [B18] The method according to any one of [B2] to [B17], wherein the recovery culture medium is a culture medium containing a basic cell culture medium to which growth factors have been added. [B19] The method according to any one of [B2] to [B18], wherein the recovery culture medium is a basic cell culture medium to which growth factors have been added. [B20] The method according to any one of [B2] to [B19], wherein the recovery culture medium has the same composition as the growth culture medium.

[0099] [B21] The method according to any one of [B2] to [B20], wherein the recovery culture medium does not contain laminin fragments or their variants. [B22] The method according to any one of [B2] to [B21], wherein the culture in the recovery culture medium is carried out for 0.5 to 10 days, preferably 2 to 5 days. [B23] The method according to any one of [B2] to [B22], wherein the cycle of culturing in the production culture medium and culturing in the recovery culture medium is repeated 2 to 10 times.

[0100] The following describes "mesenchymal stem cell populations and methods for their production," "stem cell culture supernatants and methods for their production," and "therapeutic agents containing mesenchymal stem cell populations or stem cell culture supernatants."

[0101] <4. Mesenchymal stem cell population and its production method> The inventors have newly discovered that when mesenchymal stem cells are cultured in a proliferation culture medium in the presence of laminin fragments having integrin-binding activity, according to the method described in section <1. Method for Producing Proliferating Cells>, the expression of HLA-ABC and CD105, which are cell surface markers of mesenchymal stem cells, decreases (see Example 4 below).

[0102] In this technical field, mesenchymal stem cells are known to be HLA-ABC positive and CD105 positive. Furthermore, in this technical field, positivity for cell surface markers can be expressed by the positivity rate of the cell surface markers. The positivity rate of cell surface markers can be determined by flow cytometry using a fluorescently labeled antibody for the cell surface marker, as described in the examples below.

[0103] Accordingly, according to one embodiment, a method is provided for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a proliferation culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants to obtain proliferating cells. In this method, the proliferating cells obtained have a reduced HLA-ABC positivity rate. "HLA-ABC positivity rate" represents the percentage of HLA-ABC-positive mesenchymal stem cells in the mesenchymal stem cell population.

[0104] This method makes it possible to obtain a mesenchymal stem cell population with a reduced HLA-ABC positivity rate compared to the mesenchymal stem cell population before culture. For example, this method makes it possible to obtain a mesenchymal stem cell population with an HLA-ABC positivity rate of 70% or less. In this mesenchymal stem cell population, the HLA-ABC positivity rate is preferably 60% or less, more preferably 50% or less, even more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less.

[0105] A preferred embodiment provides a method for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate and a reduced CD105 positivity rate, comprising culturing mesenchymal stem cells in a proliferation culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, to obtain proliferating cells. In this method, the proliferating cells obtained have a reduced HLA-ABC positivity rate and a reduced CD105 positivity rate. "HLA-ABC positivity rate" represents the percentage of HLA-ABC-positive mesenchymal stem cells in the mesenchymal stem cell population, and "CD105 positivity rate" represents the percentage of CD-105-positive mesenchymal stem cells in the mesenchymal stem cell population.

[0106] This method makes it possible to obtain a mesenchymal stem cell population having a reduced HLA-ABC positivity rate and a reduced CD105 positivity rate compared to the mesenchymal stem cell population before culture. For example, this method makes it possible to obtain a mesenchymal stem cell population having an HLA-ABC positivity rate of 70% or less and a CD105 positivity rate of 50% or less. In this mesenchymal stem cell population, the HLA-ABC positivity rate is preferably 60% or less, more preferably 50% or less, even more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less. In this mesenchymal stem cell population, the CD105 positivity rate is preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less.

[0107] The above-mentioned "Methchymal Stem Cell Population Production Method" can be carried out as described in section <1. Proliferating Cell Production Method>. Section <1. Proliferating Cell Production Method> states that the cell density (i.e., cell seeding density) at the start of culture should be lower than usual (0.002 to 2000 cells / cm2). However, in the above-mentioned "Methchymal Stem Cell Population Production Method," it is not necessary to adopt a lower-than-usual seeding density, and a seeding density exceeding 2000 cells / cm2 may be adopted. In other words, in the above-mentioned "Methchymal Stem Cell Population Production Method," the cell density (i.e., cell seeding density) at the start of proliferation culture can be, for example, 2001 to 1,000,000 cells / cm2, preferably 5,000 to 20,000 cells / cm2. Of course, in this method, a lower-than-usual seeding density (0.002 to 2000 cells / cm2) may also be adopted.

[0108] Mesenchymal stem cells with a low HLA-ABC positivity rate are less likely to trigger a host immune response (immune rejection) when administered as a therapeutic agent, thus having a higher cell viability and are expected to exhibit high therapeutic efficacy. Cells with a low CD105 positivity rate have high TGFb1 expression levels and suppress T cell proliferation, suggesting potential therapeutic effects on inflammatory diseases.

[0109] In this specification, the term "mesenchymal stem cells" includes both mesenchymal stem cells before culturing by the method described above and mesenchymal stem cells obtained by culturing by the method described above. That is, in this specification, the term "mesenchymal stem cells" includes not only HLA-ABC-positive mesenchymal stem cells and CD105-positive mesenchymal stem cells, but also HLA-ABC-negative mesenchymal stem cells and CD105-negative mesenchymal stem cells. Therefore, in this specification, the term "mesenchymal stem cells" can be defined as mesenchymal stem cells that are positive for CD44, CD73, and CD90, and negative for CD45, CD34, CD31, and HLA-DR.

[0110] <5. Stem cell culture supernatant and method for producing the same> The inventors have newly discovered that when mesenchymal stem cells are cultured in a proliferation culture medium in the presence of a laminin fragment having integrin-binding activity, according to the method described in section <2. Method for producing cell products>, and then cultured alternately in a production culture medium and a recovery culture medium, and the supernatant of the production culture medium is collected, the amount of cytokines, extracellular matrix, and exosomes in the culture supernatant increases with each subsequent production culture (see Example 5 below).

[0111] Therefore, according to one embodiment, a method for producing the culture supernatant of stem cells, Stem cells are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method is provided in which the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adhesion state of the cells, and the method further comprises collecting the supernatant of the production culture medium after the culture in the production culture medium. In this method, the stem cells are, for example, mesenchymal stem cells.

[0112] In this specification, the term "cell culture supernatant" refers to the supernatant obtained by culturing cells in a culture medium and removing cells and impurities from the mixture of cells and culture medium obtained after culturing; it can also be called cell-derived culture supernatant. Cell culture supernatant also includes culture supernatant that has undergone treatment such as sterilization. The terms "culture supernatant of specific cells," such as "culture supernatant of stem cells" and "culture supernatant of mesenchymal stem cells," have the same meaning.

[0113] This method can be carried out as described in section <2. Method for producing cell products>. Section <2. Method for producing cell products> states that the cell density (i.e., cell seeding density) at the start of proliferation culture should be lower than usual (0.002 to 2000 cells / cm2). However, in the above-mentioned "Method for producing culture supernatant of stem cells," it is not necessary to use a lower-than-usual seeding density, and a seeding density exceeding 2000 cells / cm2 may be used. In other words, in this method, the cell density (i.e., cell seeding density) at the start of proliferation culture can be, for example, 2001 to 1,000,000 cells / cm2, preferably 5,000 to 20,000 cells / cm2. Of course, in this method, a lower-than-usual seeding density (0.002 to 2000 cells / cm2) may also be used.

[0114] As described above, by repeatedly performing production culture and recovery culture alternately according to this method and collecting the supernatant of the production culture medium, the amounts of cytokines, extracellular matrix, and exosomes in the culture supernatant can be increased with each subsequent production culture. Therefore, in this method, it is preferable to collect the supernatant of the production culture medium after the second and subsequent production cultures in the production culture medium. It is even more preferable to collect the supernatant of the production culture medium after the third and subsequent production cultures in the production culture medium.

[0115] The supernatant of the production culture medium can be collected repeatedly as long as the secretion of cellular products such as cytokines by stem cells is maintained. In other words, there is no particular limit to the number of times production culture can be repeated in this method. For example, the supernatant of the production culture medium can be collected after the 2nd to 10th production culture cycles in the production culture medium.

[0116] According to the "Method for Producing Stem Cell Culture Supernatant" described above, it is possible to obtain stem cell culture supernatant containing large amounts of cellular products such as cytokines, extracellular matrix, and exosomes.

[0117] Specifically, according to the "Method for Producing Stem Cell Culture Supernatant" described above, a stem cell culture supernatant containing 5000 pg / mL or more of HGF (hepatocyte growth factor) can be obtained (see Figures 16 and 17). Hereinafter, this culture supernatant will also be referred to as the culture supernatant according to the first embodiment. This culture supernatant contains, for example, 5000 pg / mL or more of HGF, preferably 10000 pg / mL or more, and more preferably 15000 pg / mL or more. This culture supernatant contains, for example, 5000 to 1000000 pg / mL of HGF, preferably 10000 to 1000000 pg / mL, and more preferably 15000 to 1000000 pg / mL. HGF is known to have angiogenic and wound healing effects.

[0118] Furthermore, according to the "Method for Producing Stem Cell Culture Supernatant" described above, a stem cell culture supernatant containing 50 pg / mL or more of CD9 / CD63 EC domain fusion protein can be obtained (see Figure 42). Hereinafter, this culture supernatant will also be referred to as the culture supernatant according to the second embodiment. This culture supernatant contains, for example, 50 pg / mL or more, preferably 100 pg / mL or more, and more preferably 200 pg / mL or more of CD9 / CD63 EC domain fusion protein. This culture supernatant contains, for example, 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, and more preferably 200 to 100,000 pg / mL of CD9 / CD63 EC domain fusion protein. CD9 / CD63 EC domain fusion protein is an exosome marker protein, and a high content of CD9 / CD63 EC domain fusion protein in the culture supernatant indicates a high exosome content in the culture supernatant. Exosomes are known to have angiogenic and wound-healing effects.

[0119] In preferred embodiments, each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment may further contain 3000 pg / mL or more of MCP-1 (monocyte chemotactic protein-1), 1000 pg / mL or more of GRO (growth-related oncogene), and 5 μg / mL or more of fibronectin (see Figures 18-20). Each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment may further contain, for example, 3000 pg / mL or more, preferably 4000 pg / mL or more, and more preferably 6000 pg / mL or more of MCP-1; for example, 1000 pg / mL or more, preferably 2000 pg / mL or more, and more preferably 4000 pg / mL or more of GRO; and for example, 5 μg / mL or more, preferably 6 μg / mL or more, and more preferably 8 μg / mL or more of fibronectin. Each of the culture supernatants according to the first and second embodiments may further contain, for example, 3,000 to 1,000,000 pg / mL of MCP-1, preferably 4,000 to 1,000,000 pg / mL, more preferably 6,000 to 1,000,000 pg / mL; for example, 1,000 to 1,000,000 pg / mL of GRO, preferably 2,000 to 1,000,000 pg / mL, more preferably 4,000 to 1,000,000 pg / mL; and for example, 5 to 1,000 μg / mL of fibronectin, preferably 6 to 1,000 μg / mL, more preferably 8 to 1,000 μg / mL. MCP-1, GRO, and fibronectin are all known to have angiogenic and wound healing effects. Since GRO is also known as CXCL1, it will also be referred to as GRO / CXCL1 in this specification.

[0120] In preferred embodiments, each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment may further contain 200 pg / mL or more of TGF-1b (transforming growth factor-1b), 5 pg / mL or more of IL-4 (interleukin-4), and 10 pg / mL or more of IL-10 (interleukin-10) (see Figures 23-25). Each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment may further contain, for example, 200 pg / mL or more of TGF-1b, preferably 300 pg / mL or more, more preferably 500 pg / mL or more; for example, 5 pg / mL or more of IL-4, preferably 10 pg / mL or more, more preferably 20 pg / mL or more; and for example, 8 pg / mL or more of IL-10, preferably 10 pg / mL or more, more preferably 12 pg / mL or more. Each of the culture supernatants according to the first and second embodiments may further contain, for example, 200 to 100,000 pg / mL of TGF-1b, preferably 300 to 100,000 pg / mL, more preferably 500 to 100,000 pg / mL; IL-4, for example, 5 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 20 to 100,000 pg / mL; and IL-10, for example, 8 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 12 to 100,000 pg / mL. TGF-1b, IL-4, and IL-10 are all known to have anti-inflammatory effects.

[0121] It is preferable that each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment does not contain at least one of insulin, transferrin, and albumin. It is even more preferable that each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment does not contain any of insulin, transferrin, and albumin. In the production culture to obtain the culture supernatant, a culture medium that does not contain the above components can be used as the production culture medium to obtain a culture supernatant that does not contain the above components. Considering that the culture supernatant will be used as a therapeutic agent, it is desirable that it does not contain the above components.

[0122] Furthermore, it is preferable that the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment do not contain recombinant proteins. By using stem cells that have not been genetically modified to produce recombinant proteins, a culture supernatant free of recombinant proteins can be obtained. Considering that the culture supernatant will be used as a therapeutic agent, it is desirable that it be free of recombinant proteins, as there is a risk that unexpected components may be secreted into the culture supernatant due to the genetic manipulation of cells, and there may be unknown side effects from the recombinant proteins produced.

[0123] Furthermore, it is preferable that the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment do not contain heterologous components. Here, "heterologous" refers to a species different from the species of stem cells used to obtain the culture supernatant. Considering that the culture supernatant will be used as a therapeutic agent, it is desirable that it does not contain heterologous components.

[0124] Each of the culture supernatants according to the first and second embodiments preferably contains at least one of IL-1α (interleukin-1α), IL-1β (interleukin-1β), and TNF-α (tumor necrosis factor-α) in an amount of 0 to 15 pg / mL (see Figures 30 to 32). More preferably, each of the culture supernatants according to the first and second embodiments contains each of IL-1α, IL-1β, and TNF-α in an amount of 0 to 15 pg / mL. Since these cytokines are known to cause inflammatory symptoms, it is desirable that they are not present in large quantities in the culture supernatant.

[0125] Preferably, each of the culture supernatants according to the first embodiment and the culture supernatant according to the second embodiment contains 20 or more types of cytokines, and each cytokine has a concentration of 10 pg / mL or higher. In the examples described below, the culture supernatant collected after the third production culture contained the following cytokines: HGF, MCP-1, GRO, PDGF-AA (platelet-derived growth factor-AA), VEGF (vascular endothelial growth factor), TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, eotaxin, IL-6, G-CSF (granulocyte-colony stimulating factor), GM-CSF (granulocyte macrophage-colony stimulating factor), MCP-3 (monocyte chemotactic protein-3), IL-12P40 (40kDa subunit of interleukin-12), IP-10 (interferon gamma-induced protein-10), and MIP-1α (macrophage inflammation It contains protein-1α, and it has been demonstrated that each of these cytokines has a concentration of 10 pg / mL or higher (see Figures 16-19, 21-29, 33-41). The concentration of each of the more than 20 cytokines varies depending on the type of cytokine, but is, for example, 1,000,000 pg / mL or lower.

[0126] The culture supernatant of the first embodiment and the culture supernatant of the second embodiment are preferably culture supernatants of mesenchymal stem cells. More preferably, the culture supernatants of the first embodiment and the culture supernatants of the second embodiment are culture supernatants of umbilical cord-derived mesenchymal stem cells. Culture supernatants of mesenchymal stem cells can contain larger amounts of cytokines, extracellular matrix, and exosomes. Culture supernatants of umbilical cord-derived mesenchymal stem cells can contain particularly large amounts of cytokines, extracellular matrix, and exosomes.

[0127] The embodiments described later describe the culture supernatant of mesenchymal stem cells. However, it is known that other stem cells, such as pluripotent stem cells like ES cells and iPS cells, and somatic stem cells like neural stem cells, skin stem cells, liver stem cells, muscle stem cells, and adipose stem cells, also secrete cytokines, extracellular matrix, and exosomes into the culture medium. Therefore, even if the above-described "method for producing culture supernatant of stem cells" is performed using other stem cells, such as pluripotent stem cells like ES cells and iPS cells, or somatic stem cells like neural stem cells, skin stem cells, liver stem cells, muscle stem cells, and adipose stem cells, a culture supernatant containing large amounts of cytokines, extracellular matrix, and exosomes can be obtained, similar to the "culture supernatant of mesenchymal stem cells."

[0128] Furthermore, when the above-described "method for producing culture supernatant of stem cells" is performed using cells other than stem cells that are known to secrete cytokines, extracellular matrix, and exosomes into the culture medium, a culture supernatant containing large amounts of cytokines, extracellular matrix, and exosomes can be obtained, similar to the "culture supernatant of stem cells." Therefore, the above-described "method for producing culture supernatant of stem cells" can be extended to "method for producing culture supernatant of cells," and "culture supernatant of stem cells" can be extended to "culture supernatant of cells."

[0129] <6. Therapeutic Agents> The inventors have newly discovered that when mesenchymal stem cells are cultured in a proliferation culture medium in the presence of a laminin fragment having integrin-binding activity, according to the method described in section <1. Method for Producing Proliferating Cells>, the resulting mesenchymal stem cells can significantly increase blood flow in the ischemic lower limbs of rats in a lower limb ischemia model (see Example 6 below).

[0130] Therefore, in another respect, a therapeutic agent comprising the mesenchymal stem cell population of the present invention is provided. As described above, the mesenchymal stem cell population of the present invention can be obtained by culturing mesenchymal stem cells in a proliferation culture medium in the presence of a laminin fragment having integrin-binding activity, according to the method described in section <1. Method for producing proliferating cells>. As described above, the mesenchymal stem cell population of the present invention differs from conventional knowledge regarding mesenchymal stem cell populations in that it has a low positivity rate for specific cell surface markers HLA-ABC.

[0131] Furthermore, from another perspective, a therapeutic agent containing the culture supernatant of the present invention is provided. As described above, the culture supernatant of the present invention can be obtained by culturing mesenchymal stem cells in a proliferation culture medium in the presence of a laminin fragment having integrin binding activity, following the method described in section <2. Method for producing cell products>, then culturing in a production culture medium, and collecting the supernatant of the production culture medium. Alternatively, the culture supernatant of the present invention can be obtained by culturing mesenchymal stem cells in a proliferation culture medium in the presence of a laminin fragment having integrin binding activity, following the method described in section <2. Method for producing cell products>, then repeatedly culturing in a production culture medium and a recovery culture medium, and collecting the supernatant of the production culture medium. As described above, the culture supernatant of the present invention is characterized by containing large amounts of cytokines, extracellular matrix, and exosomes compared to conventional culture supernatants.

[0132] The mesenchymal stem cell population of the present invention has been demonstrated to secrete cytokines (e.g., HGF, MCP-1, GRO / CXCL1, PDGF-AA, VEGF, TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, EOTAXIN, IL-6, G-CSF, GM-CSF, MCP-3, IL-12P40, IP-10, and MIP-1α), extracellular matrix (e.g., Fibronectin), and exosomes, and the culture supernatant of the present invention has been demonstrated to contain cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells. Therefore, the mesenchymal stem cell population or the therapeutic agent comprising the culture supernatant of the present invention can be used to treat diseases in which mesenchymal stem cells are known to exert therapeutic effects, diseases in which the culture supernatant of mesenchymal stem cells is known to exert therapeutic effects, as well as diseases in which cytokines are known to exert therapeutic effects, diseases in which extracellular matrix is ​​known to exert therapeutic effects, and diseases in which exosomes are known to exert therapeutic effects.

[0133] For example, the mesenchymal stem cell population or the therapeutic agent containing the culture supernatant of the present invention can be used for the treatment of ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion; wounds such as epithelial wounds and burns; and sarcopenia associated with aging, since HGF, MCP-1, MCP-3, GRO / CXCL1, Fibronectin, PDGF-AA, VEGF, IL-8, EOTAXIN, IL-6, IP-10, and exosomes are known to have angiogenic effects.

[0134] Furthermore, the mesenchymal stem cell population or the therapeutic agent containing the culture supernatant of the present invention can be used to treat arthritis such as rheumatoid arthritis, herniated discs, and osteoarthritis; inflammatory diseases such as nephritis, keratitis, and cytokine storms; and mental disorders such as autism and insomnia, which are thought to be partly caused by neuroinflammation, because TGF-1b, IL-4, IL-10, and IL-13 are known to have anti-inflammatory effects.

[0135] Furthermore, since IL-7, IL-15, GM-CSF, and G-CSF are thought to be involved in immunomodulation, therapeutic agents containing the mesenchymal stem cell population or culture supernatant of the present invention can be used to treat immune diseases such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen diseases, multiple sclerosis, and autoimmune diseases; as well as cancer diseases.

[0136] When a population of mesenchymal stem cells is used as the active ingredient in a therapeutic agent, the mesenchymal stem cells of the present invention can continuously secrete cell products such as cytokines, thus allowing for the expectation of long-term therapeutic effects. On the other hand, when the culture supernatant of mesenchymal stem cells is used as the active ingredient in a therapeutic agent, the culture supernatant of the present invention contains a large amount of cell products such as cytokines, thus allowing for the expectation of sufficient therapeutic effects.

[0137] The therapeutic agent containing the mesenchymal stem cell population of the present invention and the therapeutic agent containing the culture supernatant of the present invention (hereinafter collectively referred to as the therapeutic agent) are, for example, liquid formulations, preferably injectable or topical formulations. The therapeutic agent may be diluted with a pharmaceutically acceptable medium. A pharmaceutically acceptable medium is, for example, a mesenchymal stem cell culture medium or infusion preparation. The therapeutic agent may contain additives to increase storage stability, isotonicity, absorption, and / or viscosity. Alternatively, if the therapeutic agent contains a mesenchymal stem cell population, it may be in the form of a cell sheet.

[0138] The dosage of the therapeutic agent can be appropriately determined according to the target disease, age, weight, and symptoms. If the therapeutic agent contains a population of mesenchymal stem cells, the single dose of the therapeutic agent is, for example, 1,000 to 1,000,000,000 cells / kg, preferably 1,000,000 to 1,000,000,000 cells / kg. This dose may be administered multiple times as a single dose, or this dose may be divided into multiple doses. If the therapeutic agent contains culture supernatant, the single dose of the therapeutic agent, expressed in terms of the volume of culture supernatant before dilution (mL), is, for example, 0.01 to 100 mL / kg, preferably 0.1 to 10 mL / kg. This dose may be administered multiple times as a single dose, or this dose may be divided into multiple doses.

[0139] The method of administering the therapeutic agent is not particularly limited, but examples include intravenous injection, intra-arterial injection, subcutaneous injection, intra-lymph node injection, intraperitoneal injection, application of the liquid to tissue, attachment of cell sheets, or direct injection or direct transplantation to the local area.

[0140] <7. Preferred Embodiments> Preferred embodiments of the present invention are summarized below. <7-1. Mesenchymal stem cell population and method of production thereof> [C1] A method for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a proliferation culture medium in the presence of a culture substrate selected from laminin fragments having integrin binding activity and their variants, to obtain proliferating cells. [C2] The method according to [C1], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, or umbilical cord blood-derived mesenchymal stem cells. [C3] The method according to [C1] or [C2], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells, preferably umbilical cord-derived mesenchymal stem cells. [C4] The method according to [C1], wherein the mesenchymal stem cells are human mesenchymal stem cells. [C5] The method according to [C1] or [C2], wherein the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells.

[0141] [C6] The method according to any one of [C1] to [C3], wherein the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells or human adipose-derived mesenchymal stem cells, preferably human umbilical cord-derived mesenchymal stem cells. [C7] The method according to any one of [C1] to [C6], wherein the growth culture medium is a growth culture medium containing a protein that promotes the proliferation of the stem cells. [C8] The method according to any one of [C1] to [C7], wherein the growth culture medium is a culture medium containing a basic cell culture medium to which growth factors have been added. [C9] The method according to any one of [C1] to [C8], wherein the growth culture medium is a basic cell culture medium to which growth factors have been added. [C10] The method according to any one of [C1] to [C9], wherein the culture is carried out until the cells reach a confluent state.

[0142] [C11] The method according to any one of [C1] to [C10], wherein the culture is performed in a culture vessel having a bottom area of ​​500 cm2 or more. [C12] The method according to any one of [C1] to [C11], wherein the culture is carried out in a culture vessel having a bottom area of ​​500 to 10000 cm2. [C13] The method according to any one of [C1] to [C12], wherein the culture substrate is a laminin fragment having integrin binding activity. [C14] The method according to [C13], wherein the laminin fragment is a human-derived laminin fragment. [C15] The method according to [C13] or [C14], wherein the laminin fragment is a laminin E8 fragment.

[0143] [C16] The method according to any one of [C13] to [C15], wherein the laminin fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [C17] The method according to any one of [C13] to [C16], wherein the laminin fragment is a laminin 511 E8 fragment. [C18] The method according to any one of [C1] to [C12], wherein the culture substrate is a modified laminin fragment having integrin-binding activity. [C19] The method according to [C18], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and another functional molecule. [C20] The method according to [C18] or [C19], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule.

[0144] [C21] The method according to [C19] or [C20], wherein the laminin fragment is a laminin E8 fragment. [C22] The method according to [C21], wherein the laminin E8 fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [C23] The method according to [C21] or [C22], wherein the laminin E8 fragment is a laminin 511 E8 fragment. [C24] The method according to [C21] or [C22], wherein the laminin E8 fragment is a laminin 421 E8 fragment. [C25] The method according to any one of [C20] to [C24], wherein the growth factor binding molecule is heparan sulfate.

[0145] [C26] The method according to any one of [C18] to [C25], wherein the modified product is a complex of laminin 511 E8 fragment and heparan sulfate. [C27] The method according to any one of [C18] to [C25], wherein the modified product is a complex of laminin 421 E8 fragment and heparan sulfate. [C28] The method according to any one of [C1] to [C27], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.005 μg to 2 μg per 1 cm² of culture area of ​​the culture vessel. [C29] The method according to any one of [C1] to [C28], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.01 μg to 0.5 μg per 1 cm² of culture area of ​​the culture vessel. [C30] The method according to any one of [C1] to [C29], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.05 μg to 0.25 μg per 1 cm² of culture area of ​​the culture vessel. [C31] The method according to any one of [C1] to [C30], wherein the reduced HLA-ABC positivity rate is 70% or less, preferably 60% or less, more preferably 50% or less, even more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less.

[0146] [D1] A mesenchymal stem cell population in which the proportion of HLA-ABC positive mesenchymal stem cells is 70% or less. [D2] The mesenchymal stem cell population according to [D1], wherein the proportion of HLA-ABC positive mesenchymal stem cells is 60% or less, preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less. [D3] The mesenchymal stem cell population described in [D1] or [D2], wherein the proportion of CD105-positive mesenchymal stem cells is 50% or less. [D4] The mesenchymal stem cell population according to [D3], wherein the proportion of CD105-positive mesenchymal stem cells is 40% or less, preferably 30% or less, more preferably 20% or less, and even more preferably 10% or less. A population of mesenchymal stem cells obtained by any one of the methods described in [D5] [C1]~[C31]. [D6] The mesenchymal stem cell population described in any one of [D1] to [D4], obtained by the method described in any one of [C1] to [C31].

[0147] <7-2. Stem cell culture supernatant and method for producing the same> [E1] A method for producing a culture supernatant of stem cells, Stem cells are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. After culturing in the production culture medium, the proliferating cells are cultured in the recovery culture medium while maintaining the adherent state. Includes, A method comprising the alternating repetition of the culturing in the production culture medium and the culturing in the recovery culture medium while maintaining the adhesion state of the cells, wherein the method further includes collecting the supernatant of the production culture medium after the culturing in the production culture medium. [E2] The method according to [E1], wherein the supernatant of the production culture medium is collected after the second or subsequent cultures in the production culture medium, preferably after the third or subsequent cultures in the production culture medium. [E3] The method according to [E1] or [E2], wherein the stem cells are somatic stem cells such as mesenchymal stem cells, neural stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells; or pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). [E4] The method according to any one of [E1] to [E3], wherein the stem cells are mesenchymal stem cells. [E5] The method according to any one of [E1] to [E4], wherein the stem cells are umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, or umbilical cord blood-derived mesenchymal stem cells.

[0148] [E6] The method according to any one of [E1] to [E5], wherein the stem cells are umbilical cord-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells, preferably umbilical cord-derived mesenchymal stem cells. [E7] The method according to [E1] or [E2], wherein the stem cells are human stem cells. [E8] The method according to any one of [E1] to [E3], wherein the stem cells are human somatic stem cells such as human mesenchymal stem cells, human neural stem cells, human skin stem cells, human liver stem cells, human muscle stem cells, and human adipose stem cells; or human pluripotent stem cells such as human induced pluripotent stem cells (human iPS cells) and human embryonic stem cells (human ES cells). [E9] The method according to any one of [E1] to [E4], wherein the stem cells are human mesenchymal stem cells. [E10] The method according to any one of [E1] to [E5], wherein the stem cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells.

[0149] [E11] The method according to any one of [E1] to [E6], wherein the stem cells are human umbilical cord-derived mesenchymal stem cells or human adipose-derived mesenchymal stem cells, preferably human umbilical cord-derived mesenchymal stem cells. [E12] The method according to any one of [E1] to [E11], wherein the growth culture medium is a growth culture medium containing a protein that promotes the proliferation of the stem cells. [E13] The method according to any one of [E1] to [E12], wherein the growth culture medium is a culture medium containing a basic cell culture medium to which growth factors have been added. [E14] The method according to any one of [E1] to [E13], wherein the growth medium is a basic cell culture medium to which growth factors have been added. [E15] The method according to any one of [E1] to [E14], wherein the culture in the growth medium is carried out until the cells reach a confluent state.

[0150] [E16] The method according to any one of [E1] to [E15], wherein the culture in the growth medium is carried out in a culture vessel having a bottom area of ​​500 cm2 or more. [E17] The method according to any one of [E1] to [E16], wherein the culture in the growth medium is carried out in a culture vessel having a bottom area of ​​500 to 10000 cm2. [E18] The method according to any one of [E1] to [E17], wherein the culture substrate is a laminin fragment having integrin binding activity. [E19] The method according to [E18], wherein the laminin fragment is a human-derived laminin fragment. [E20] The method according to [E18] or [E19], wherein the laminin fragment is a laminin E8 fragment.

[0151] [E21] The method according to any one of [E18] to [E20], wherein the laminin fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [E22] The method according to any one of [E18] to [E21], wherein the laminin fragment is a laminin 511 E8 fragment. [E23] The method according to any one of [E1] to [E17], wherein the culture substrate is a modified laminin fragment having integrin-binding activity. [E24] The method according to [E23], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and another functional molecule. [E25] The method according to [E23] or [E24], wherein the modified product is a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule.

[0152] [E26] The method according to [E24] or [E25], wherein the laminin fragment is a laminin E8 fragment. [E27] The method according to [E26], wherein the laminin E8 fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. [E28] The method according to [E26] or [E27], wherein the laminin E8 fragment is a laminin 511 E8 fragment. [E29] The method according to [E26] or [E27], wherein the laminin E8 fragment is a laminin 421 E8 fragment. [E30] The method according to any one of [E25] to [E29], wherein the growth factor binding molecule is heparan sulfate.

[0153] [E31] The method according to any one of [E23] to [E30], wherein the modified product is a complex of laminin 511 E8 fragment and heparan sulfate. [E32] The method according to any one of [E23] to [E30], wherein the modified product is a complex of laminin 421 E8 fragment and heparan sulfate. [E33] The method according to any one of [E1] to [E32], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.005 μg to 2 μg per 1 cm² of culture area of ​​the culture vessel. [E34] The method according to any one of [E1] to [E33], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.01 μg to 0.5 μg per 1 cm² of culture area of ​​the culture vessel. [E35] The method according to any one of [E1] to [E34], wherein the concentration of the laminin fragment or its variant in the growth culture medium is 0.05 μg to 0.25 μg per 1 cm² of culture area of ​​the culture vessel.

[0154] [E36] The method according to any one of [E1] to [E35], wherein the production culture medium is a culture medium that does not contain heterologous components. [E37] The method according to any one of [E1] to [E36], wherein the production culture medium is a culture medium that does not contain cytokines or insulin. [E38] The method according to any one of [E1] to [E37], wherein the production culture medium is a culture medium that does not contain protein. [E39] The method according to any one of [E1] to [E38], wherein the production culture medium is a serum-free culture medium. [E40] The method according to any one of [E1] to [E39], wherein the production culture medium is a culture medium containing a basic cell culture medium, or a culture medium containing a basic cell culture medium to which cell nutrients have been added.

[0155] [E41] The method according to any one of [E1] to [E40], wherein the production culture medium is a basic cell culture medium or a basic cell culture medium to which cell nutrients have been added. [E42] The method according to any one of [E1] to [E41], wherein the production culture medium is a basic cell culture medium to which cell nutrients, preferably amino acids, have been added. [E43] The method according to any one of [E1] to [E42], wherein the production culture medium does not contain laminin fragments or their variants. [E44] The method according to any one of [E1] to [E43], wherein the culture in the production culture medium is carried out for 0.5 to 10 days, preferably 2 to 5 days. [E45] The method according to any one of [E1] to [E44], wherein the recovery culture medium is the proliferation culture medium of the stem cells.

[0156] [E46] The method according to any one of [E1] to [E45], wherein the recovery culture medium is a culture medium containing a protein that promotes the proliferation of the stem cells. [E47] The method according to any one of [E1] to [E46], wherein the recovery culture medium is a culture medium containing a basic cell culture medium to which growth factors have been added. [E48] The method according to any one of [E1] to [E47], wherein the recovery culture medium is a basic cell culture medium to which growth factors have been added. [E49] The method according to any one of [E1] to [E48], wherein the recovery culture medium has the same composition as the growth culture medium. [E50] The method according to any one of [E1] to [E49], wherein the recovery culture medium does not contain laminin fragments or their variants. [E51] The method according to any one of [E1] to [E50], wherein the culture in the recovery culture medium is carried out for 0.5 to 10 days, preferably 2 to 5 days. [E52] The method according to any one of [E1] to [E51], wherein the cycle of culturing in the production culture medium and culturing in the recovery culture medium is repeated 2 to 10 times.

[0157] [F1] Stem cell culture supernatant containing 5000 pg / mL or more of HGF. [F2] The culture supernatant according to [F1], wherein the culture supernatant contains 10,000 pg / mL or more, preferably 15,000 pg / mL or more of HGF. [F3] The culture supernatant according to [F1] or [F2], wherein the culture supernatant contains 5,000 to 1,000,000 pg / mL, preferably 10,000 to 1,000,000 pg / mL, and more preferably 15,000 to 1,000,000 pg / mL of HGF. [F4] Stem cell culture supernatant containing CD9 / CD63 EC domain fusion protein at a concentration of 50 pg / mL or higher. [F5] The culture supernatant according to [F4], wherein the culture supernatant contains 100 pg / mL or more, preferably 200 pg / mL or more, of the CD9 / CD63 EC domain fusion protein.

[0158] [F6] The culture supernatant according to [F4] or [F5], wherein the culture supernatant contains a CD9 / CD63 EC domain fusion protein in a concentration of 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, more preferably 200 to 100,000 pg / mL. [F7] The culture supernatant according to any one of [F1] to [F3], wherein the culture supernatant further contains 50 pg / mL or more of CD9 / CD63 EC domain fusion protein. [F8] The culture supernatant according to any one of [F1] to [F3], wherein the culture supernatant further contains 100 pg / mL or more, preferably 200 pg / mL or more, of CD9 / CD63 EC domain fusion protein. [F9] The culture supernatant according to any one of [F1] to [F3], wherein the culture supernatant further contains a CD9 / CD63 EC domain fusion protein in a concentration of 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, more preferably 200 to 100,000 pg / mL. [F10] The culture supernatant according to any one of [F1] to [F9], wherein the culture supernatant further contains 3000 pg / mL or more of MCP-1, 1000 pg / mL or more of GRO, and 5 μg / mL or more of fibronectin.

[0159] [F11] The culture supernatant according to any one of [F1] to [F9], wherein the culture supernatant further contains MCP-1 in an amount of 4000 pg / mL or more, preferably 6000 pg / mL or more; GRO in an amount of 2000 pg / mL or more, preferably 4000 pg / mL or more; and fibronectin in an amount of 6 μg / mL or more, preferably 8 μg / mL or more. [F12] The culture supernatant according to any one of [F1] to [F9], wherein the culture supernatant further contains 3,000 to 1,000,000 pg / mL, preferably 4,000 to 1,000,000 pg / mL of MCP-1; 1,000 to 1,000,000 pg / mL, preferably 2,000 to 1,000,000 pg / mL of GRO; and 5 to 1,000 μg / mL, preferably 6 to 1,000 μg / mL of fibronectin. [F13] The culture supernatant according to any one of [F1] to [F12], wherein the culture supernatant further contains 200 pg / mL or more of TGF-1b, 5 pg / mL or more of IL-4, and 8 pg / mL or more of IL-10. [F14] The culture supernatant according to any one of [F1] to [F12], wherein the culture supernatant further contains 300 pg / mL or more, preferably 500 pg / mL or more, TGF-1b; 10 pg / mL or more, preferably 20 pg / mL or more, IL-4; and 10 pg / mL or more, preferably 12 pg / mL or more, IL-10. [F15] The culture supernatant according to any one of [F1] to [F12], wherein the culture supernatant further contains 200 to 100,000 pg / mL, preferably 300 to 100,000 pg / mL, more preferably 500 to 100,000 pg / mL of TGF-1b; 5 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 20 to 100,000 pg / mL of IL-4; and 8 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 12 to 100,000 pg / mL of IL-10.

[0160] [F16] The culture supernatant according to any one of [F1] to [F15], wherein the culture supernatant does not contain at least one of insulin, transferrin, and albumin. [F17] The culture supernatant according to any one of [F1] to [F16], wherein the culture supernatant does not contain insulin, transferrin, or albumin. [F18] The culture supernatant according to any one of [F1] to [F17], wherein the culture supernatant does not contain recombinant protein. [F19] The culture supernatant according to any one of [F1] to [F18], wherein the culture supernatant contains at least one of IL-1α, IL-1β, and TNF-α in an amount of 0 to 15 pg / mL. [F20] The culture supernatant according to any one of [F1] to [F19], wherein the culture supernatant contains IL-1α, IL-1β, and TNF-α in amounts of 0 to 15 pg / mL each.

[0161] [F21] The culture supernatant according to any one of [F1] to [F20], wherein the culture supernatant contains 20 or more types of cytokines, and each cytokine has a concentration of 10 pg / mL or more. [F22] The culture supernatant according to any one of [F1] to [F21], wherein the culture supernatant is the culture supernatant of somatic stem cells such as mesenchymal stem cells, neural stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells; or the culture supernatant of pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). [F23] The culture supernatant according to any one of [F1] to [F22], wherein the culture supernatant is the culture supernatant of mesenchymal stem cells. [F24] The culture supernatant according to any one of [F1] to [F23], wherein the culture supernatant is the culture supernatant of umbilical cord-derived mesenchymal stem cells, the culture supernatant of bone marrow-derived mesenchymal stem cells, the culture supernatant of adipose-derived mesenchymal stem cells, the culture supernatant of placenta-derived mesenchymal stem cells, or the culture supernatant of umbilical cord blood-derived mesenchymal stem cells. [F25] The culture supernatant according to any one of [F1] to [F24], wherein the culture supernatant is the culture supernatant of umbilical cord-derived mesenchymal stem cells or the culture supernatant of adipose-derived mesenchymal stem cells, preferably the culture supernatant of umbilical cord-derived mesenchymal stem cells.

[0162] [F26] The culture supernatant according to any one of [F1] to [F23], wherein the culture supernatant is the culture supernatant of human mesenchymal stem cells. [F27] The culture supernatant according to any one of [F1] to [F24], wherein the culture supernatant is the culture supernatant of human umbilical cord-derived mesenchymal stem cells, the culture supernatant of human bone marrow-derived mesenchymal stem cells, the culture supernatant of human adipose-derived mesenchymal stem cells, the culture supernatant of human placenta-derived mesenchymal stem cells, or the culture supernatant of human umbilical cord blood-derived mesenchymal stem cells. [F28] The culture supernatant according to any one of [F1] to [F25], wherein the culture supernatant is the culture supernatant of human umbilical cord-derived mesenchymal stem cells or the culture supernatant of human adipose-derived mesenchymal stem cells, preferably the culture supernatant of human umbilical cord-derived mesenchymal stem cells. [F29] Culture supernatant obtained by any one of the methods described in [E1]~[E52]. [F30] The culture supernatant according to any one of [F1] to [F28], obtained by the method described in any one of [E1] to [E52].

[0163] <7-3. Therapeutic Agents> A therapeutic agent comprising a mesenchymal stem cell population described in any one of [G1], [D1], to [D6], or a culture supernatant described in any one of [F1], to [F30]. A therapeutic agent containing a mesenchymal stem cell population described in any one of [G2] [D1] to [D6]. A therapeutic agent containing the culture supernatant described in any one of [G3], [F1], or [F30]. [G4] The therapeutic agent described in any one of [G1] to [G3] is for the treatment of ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion; wounds such as epithelial wounds and burns; sarcopenia associated with aging; arthritis such as rheumatism, herniated disc, and osteoarthritis; inflammatory diseases such as nephritis, keratitis, and cytokine storm; mental disorders such as autism and insomnia in which neuroinflammation is thought to be one of the causes; immune diseases such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, and autoimmune diseases; or cancer. [G5] The therapeutic agent according to any one of [G1] to [G4], wherein the therapeutic agent is for treating an ischemic disease; preferably lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; more preferably lower limb ischemia.

[0164] A method for treating a disease, comprising administering to a subject a mesenchymal stem cell population described in any one of [G6] [D1] to [D6] or a culture supernatant described in any one of [F1] to [F30]. A method for treating a disease or injury, comprising administering a mesenchymal stem cell population described in any one of [G7] [D1] to [D6] to a subject. A method for treating a disease or injury, comprising administering to a subject the culture supernatant described in any one of [G8] [F1] to [F30]. [G9] The method according to any one of [G6] to [G8], wherein the injury or illness is an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; an injury such as an epithelial wound or burn; sarcopenia associated with aging; arthritis such as rheumatism, herniated disc, or osteoarthritis; inflammatory diseases such as cytokine storm, nephritis, or keratitis; mental disorders such as autism or insomnia in which neuroinflammation is thought to be one of the causes; an immune disease such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, or autoimmune disease; or a cancerous disease. [G10] The method according to any one of [G6] to [G9], wherein the injury or illness is an ischemic disease; preferably lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; more preferably lower limb ischemia. [G11] The method according to any one of [G6] to [G10], wherein the subject is a mammal, preferably a human.

[0165] [G12] Use of a mesenchymal stem cell population described in any one of [D1] to [D6] or a culture supernatant described in any one of [F1] to [F30] for the manufacture of a therapeutic agent. [G13] Use of any one of the mesenchymal stem cell populations described in [D1] to [D6] for the manufacture of therapeutic agents. [G14] Use of culture supernatant described in any one of [F1] to [F30] for the manufacture of therapeutic agents. [G15] Use as described in any one of [G12] to [G14], where the therapeutic agent is used to treat ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion; wounds such as epithelial wounds and burns; sarcopenia associated with aging; arthritis such as rheumatism, herniated disc, and osteoarthritis; inflammatory diseases such as cytokine storms, nephritis, and keratitis; mental disorders such as autism and insomnia in which neuroinflammation is thought to be one of the causes; immune diseases such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, and autoimmune diseases; or cancer. [G16] The use according to any one of [G12] to [G15], wherein the therapeutic agent is for the treatment of an ischemic disease; preferably lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; more preferably lower limb ischemia. [Examples] [Examples]

[0166] Growth culture (1) Method In Example 1, stem cell proliferation culture was performed. As stem cells, umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in the cryopreservation solution Stem Cell Banker (Zenoac) were used.

[0167] Experiment 1-1 (Control) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) at cell counts of 5 x 10 e3 (seeding density 10 cells / cm2), 5 x 10 e4 (seeding density 100 cells / cm2), 5 x 10 e5 (seeding density 1000 cells / cm2), and 5 x 10 e6 (seeding density 10000 cells / cm2). The resulting cell suspensions were seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). For each condition, four flasks were used for seeding.

[0168] During the growth culture period, the culture medium was changed every 5 days. Five days after seeding (day 5), ten days after seeding (day 10), fifteen days after seeding (day 15), and twenty days after seeding (day 20), each flask was treated with TrypLE™ Select (Thermo Fisher Scientific) for 10-20 minutes to disperse the cells into single cells, and then the cell count was measured using a cell counter.

[0169] The number of proliferating cells is shown in Figure 1. In Figure 1, the vertical axis of the graph shows the measured number of cells divided by the flask area. Figure 2 shows a micrograph of cells cultured for 20 days at a seeding density of 10 cells / cm2.

[0170] Experiment 1-2 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) at cell counts of 5 x 10 e3 (seeding density 10 cells / cm2), 5 x 10 e4 (seeding density 100 cells / cm2), 5 x 10 e5 (seeding density 1000 cells / cm2), and 5 x 10 e6 (seeding density 10000 cells / cm2). 50 μl / 100 ml of iMatrix-511 laminin fragment (Nippi Corporation) was added, and the resulting cell suspensions were seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). For each condition, seeding was performed in four flasks.

[0171] During the growth culture period, the culture medium was changed every 5 days. Five days after sowing, iMatrix-511 laminin fragment was added again at a rate of 50 μl / 100 ml. Laminin fragment was not added at 10, 15, or 20 days after sowing.

[0172] Five days (day 5), ten days (day 10), fifteen days (day 15), and twenty days (day 20) after sowing, each flask was treated with TrypLE™ Select (Thermo Fisher Scientific) for 10-20 minutes to disperse the cells into single cells, and then the number of cells was measured using a cell counter.

[0173] The number of proliferating cells is shown in Figure 3. In Figure 3, the vertical axis of the graph shows the measured number of cells divided by the flask area. Figure 4 shows a micrograph of cells cultured for 20 days at a seeding density of 10 cells / cm2.

[0174] Experiment 1-3 (Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended at 5×10³ cells (seeding density: 10 cells / cm²), 5×10⁴ cells (seeding density: 100 cells / cm²), 5×10⁵ cells (seeding density: 1000 cells / cm²), and 5×10⁶ cells (seeding density: 10000 cells / cm²) respectively in 100 mL of MSC Expansion XSFM B medium (FUJIFILM Wako Pure Chemical Corporation), and seeded into pre-gelatin-coated peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). Gelatin coating was performed by treatment with a gelatin solution (StemSure 0.1 w / v% Gelatin Solution, FUJIFILM Wako Pure Chemical Corporation) for 2 hours.

[0175] During proliferation culture, medium exchange was performed every 5 days. At 5 days (day 5), 10 days (day 10), 15 days (day 15), and 20 days (day 20) after seeding, each flask was treated with TrypLE™ Select (Thermo Fisher Scientific) for 10 to 20 minutes to disperse into single cells, and then the number of cells was counted with a cell counter.

[0176] The number of proliferated cells is shown in Figure 5. In Figure 5, the vertical axis of the graph represents the value obtained by dividing the counted number of cells by the area of the flask. Furthermore, a micrograph of cells after 20 days of culture at a seeding density of 10 cells / cm² is shown in Figure 6.

[0177] (2) Results When umbilical cord-derived mesenchymal stem cells (UCMSCs) proliferated and reached a confluent state in the T512 flasks, approximately 3×10⁷ cells (i.e., approximately 60000 cells / cm²) were recovered. Accordingly, in Figures 1, 3 and 5, approximately 60000 cells / cm² represents the confluent state.

[0178] The results in Figure 1 show that, in the absence of laminin fragments, when stem cells are seeded and cultured at a seeding density of 10,000 cells / cm2, the cells can proliferate to a confluent state. However, when stem cells are seeded and cultured at a low cell density of 1,000 cells / cm2 or less, cell proliferation stops, and they cannot proliferate to a confluent state. Furthermore, the micrographs in Figure 2 show that, in the absence of laminin fragments, when stem cells are seeded and cultured at a low cell density of 10 cells / cm2, the cells do not proliferate to a confluent state and proliferation stops at a colony state.

[0179] The results in Figure 3 show that, in the presence of laminin fragments, stem cells can be cultured at low cell densities of 1000 cells / cm2 or less and still proliferate to a confluent state. Furthermore, the micrographs in Figure 4 show that, in the presence of laminin fragments, stem cells can be cultured at low cell densities of 10 cells / cm2 and still proliferate to a confluent state.

[0180] Furthermore, the inventors have demonstrated that umbilical cord-derived mesenchymal stem cells (UCMSCs), when seeded at a cell density of 2 cells / cm2 and cultured in a growth medium containing iMatrix-511 laminin fragment, can also proliferate to a confluent state, following the same method as in Experiments 1-2.

[0181] The results in Figure 5 show that, in the presence of gelatin, when stem cells are seeded at a seeding density of 10,000 cells / cm2 and cultured, the cells can proliferate to a confluent state. However, when stem cells are seeded at a low cell density of 1,000 cells / cm2 or less and cultured, cell proliferation stops, and they cannot proliferate to a confluent state. Furthermore, the micrograph in Figure 6 shows that, in the presence of gelatin, when stem cells are seeded at a low cell density of 10 cells / cm2 and cultured, the cells do not proliferate to a confluent state and proliferation stops at a colony state.

[0182] These results indicate that culturing stem cells in the presence of laminin fragments allows even stem cells seeded at low cell densities to proliferate to a confluent state. [Examples]

[0183] Production culture (1) Method In Example 2, stem cell proliferation culture was performed, followed by production culture. As stem cells, umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in the cryopreservation solution Stem Cell Banker (Zenoac) were used.

[0184] Experiment 2-1 (Comparative Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended at a cell count of 5 x 10 e5 (seeding density 1000 cells / cm2) in 100 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B), and the resulting cell suspension was seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.).

[0185] During the growth culture, the culture medium was changed every 5 days. Fifteen days after seeding (day 15), the medium was replaced twice with PBS to wash away any remaining medium, and then 120 ml of a protein-free medium (hereinafter also referred to as MSC medium A) based on DMEM / F12 medium with added amino acids was added. The added amino acids were MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The cells were cultured for 3 days (production culture) after adding MSC medium A. After 3 days of culture, two images of the cells were taken using an Olympus microscope. The two images were taken at different positions in the flask. The micrographs are shown in Figures 7A and 7B.

[0186] Experiment 2-2 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended at a cell count of 5 x 10 e5 (seeding density 1000 cells / cm2) in 100 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Corporation) (hereinafter also referred to as MSC medium B). 50 μl / 100 ml of iMatrix-511 laminin fragment (Nippi Corporation) was added, and the resulting cell suspension was seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.).

[0187] During the growth culture period, the culture medium was changed every 5 days. Five days after seeding, iMatrix-511 laminin fragment was added again at a rate of 50 μl / 100 ml. Laminin fragment was not added 10 days after seeding.

[0188] Fifteen days after seeding (day 15), the culture medium was replaced twice with PBS to wash away any remaining medium. Then, 120 ml of a protein-free medium (hereinafter also referred to as MSC medium A) based on DMEM / F12 medium with added amino acids was added. The added amino acids were MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.). Cells were cultured for 3 days (production culture) after adding MSC medium A. After 3 days of culture, two images of the cells were taken using an Olympus microscope. The two images were taken at different positions within the flask. The micrographs are shown in Figures 8A and 8B.

[0189] (2) Results In Figure 7A, some cells had detached and formed cell clumps. The locations of the cell clumps in Figure 7A are indicated by arrows. In Figure 7B, most of the cells had detached and were no longer present. On the other hand, in both Figure 8A and Figure 8B, the cells remained attached and had not detached.

[0190] From these results, it can be seen that when stem cells are cultured and proliferated in the presence of a laminin fragment, and the obtained proliferated cells are then cultured in a protein-free culture solution containing no exogenous components, the adherent state of the proliferated cells can be maintained without the cells detaching from the culture vessel during the culture. Examples

[0191] Long-term production culture (1) Method In Example 3, after proliferation culture of stem cells was performed, long-term production culture was carried out. The culture step performed in Example 3 is schematically shown in Figure 9. Umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in Stem Cell Banker (Zenoac) cryopreservation solution were used as the stem cells.

[0192] Experiment 3-1 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended at a cell count of 5×105 (seeding density: 1000 cells / cm2) in 100 mL of MSC Expansion XSFM B (FUJIFILM Wako Pure Chemical Corporation) (hereinafter also referred to as MSC medium B), iMatrix-511 laminin fragment (Nippi Co., Ltd.) was added at 50 μL / 100 mL, and the resulting cell suspension was seeded into a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).

[0193] During proliferation culture, medium exchange was performed every 5 days. Five days after seeding, the iMatrix-511 laminin fragment was added again at 50 μL / 100 mL. No laminin fragment was added 10 days after seeding.

[0194] Fifteen days after seeding (day 15), the cells were confirmed to have reached confluence, and the culture medium was replaced twice with PBS to wash away any remaining medium. Then, 120 ml of a protein-free medium (hereinafter referred to as MSC medium A) based on DMEM / F12 medium with added amino acids was added. The added amino acids were MEM essential amino acid solution (Fujifilm Wako Pure Chemical Corporation) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Corporation). The cells were cultured for 4 days after the addition of MSC medium A. This culture is represented as "first production culture" in Figure 9.

[0195] After the first production culture, the culture supernatant of MSC medium A was collected. Cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems). Three types of cytokines were analyzed: vascular endothelial growth factor (VEGF), interleukin-7 (IL-7), and hepatocyte growth factor (HGF).

[0196] Furthermore, after the first production culture, the culture was returned to MSC medium B and cultured for 3 days (recovery culture). After the 3 days of recovery culture, the culture was again replaced with MSC medium A and cultured for 4 days. This culture is represented as "second production culture" in Figure 9.

[0197] After the second production culture, the culture supernatant of MSC medium A was collected. Cytokines (VEGF, IL-7, and HGF) contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).

[0198] Furthermore, after the second production culture, the culture was returned to MSC medium B and cultured for 3 days (recovery culture). After the 3 days of recovery culture, the culture was again replaced with MSC medium A and cultured for 4 days. This culture is represented as the "third production culture" in Figure 9.

[0199] After the third production culture cycle, the culture supernatant of MSC medium A was collected. Cytokines (VEGF, IL-7, and HGF) contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).

[0200] As described above, we performed a total of three production culture cycles, and carried out a total of three culture supernatant collections and cytokine analyses.

[0201] Experiment 3-2 (Comparative Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended in 20 ml of MSCExpansion XSFM B (Fujifilm Wako Pure Chemical Corporation) (MSC medium B) at a cell count of 1.5 x 10 e5 (seeding density 1000 cells / cm2) and seeded in a T150 flask (Corning) without the addition of iMatrix-511 laminin fragment. Growth culture was performed using the same procedure as in Experiment 3-1, except that laminin fragment was not added.

[0202] Fifteen days after seeding (day 15), the cells were confirmed to have reached confluence, and were replaced with MSC medium A, as in Experiment 3-1. After two days of incubation, the culture supernatant of MSC medium A was collected. Cytokines (VEGF, IL-7, and HGF) in the culture supernatant were analyzed using an ELISA kit (R&D Systems).

[0203] In this experiment, since no laminin fragments were added, cells began to detach from the culture vessel 3 days after switching to MSC medium A. Therefore, the culture supernatant was collected 2 days after switching to MSC medium A.

[0204] (2) Results The results of cytokine quantification are shown in Figures 10-12. The graphs in Figures 10-12, from left to right, are as follows: In an example of the present invention, the amount of cytokines in the culture supernatant recovered after the first production culture, In an example of the present invention, the amount of cytokines in the culture supernatant recovered after the second production culture, In the example of the present invention, the amount of cytokines in the culture supernatant recovered after the third production culture, and Amount of cytokines in the culture supernatant recovered in the comparative example This indicates.

[0205] The results shown in Figures 10-12 demonstrate that long-term production culture according to the present invention can produce cytokines in amounts equal to or greater than those of the comparative example, and that these production amounts do not decrease or even increase with repeated production cultures. VEGF and HGF are cytokines that have angiogenic effects. While the production amount of VEGF was sometimes slightly lower than that of the comparative example, the absolute amount of VEGF produced was large, so it can be said that it was produced in sufficient quantities. HGF showed a tendency for production to increase with repeated production cultures. In addition, IL-7 is a cytokine involved in the activation of immune cells and inflammation. IL-7 also showed a tendency for production to increase with repeated production cultures.

[0206] In Example 3, three production cultures were performed, and the culture supernatant was collected and cytokine analysis was conducted three times. However, the inventors demonstrated that cytokine production is possible by performing a total of six production cultures.

[0207] These results indicate the following: When stem cells are cultured and proliferated in the presence of laminin fragments, and the resulting proliferated cells are then cultured in a protein-free culture medium that does not contain foreign components, production culture can be performed while maintaining the adhesion state of the proliferated cells. Furthermore, after this production culture, recovery culture can be performed while maintaining the adhesion state of the cells, restoring the cells' ability to produce cellular products. This allows for repeated production cultures while maintaining the adhesion state of the cells, and as a result, large quantities of cellular products can be produced over a long period using a simple method. [Examples]

[0208] Mesenchymal stem cell population In Example 4, mesenchymal stem cells were cultured in the presence of laminin fragments, and the positivity rate of cell surface markers in the resulting mesenchymal stem cell population was analyzed. Furthermore, the expression of cell surface markers in the resulting mesenchymal stem cell population, obtained by culture of mesenchymal stem cells in the presence of laminin fragments, was confirmed by immunohistochemical staining.

[0209] (1) Method Experiment 4-1 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) or adipose-derived mesenchymal stem cells (ADMSCs) were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) at a cell count of 5 x 10 e5 (seeding density 1000 cells / cm2). 50 μl / 100 ml of iMatrix-511 laminin fragment (Nippi Corporation) was added, and the resulting cell suspension was seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). Umbilical cord-derived mesenchymal stem cells were cultured for 1, 2, and 3 weeks, while adipose-derived mesenchymal stem cells were cultured for 1 and 2 weeks.

[0210] After culturing, cells were detached by treatment with TrypLE Select solution (Thermo Fisher Scientific). The obtained cells were stained with PE (phycoerythrin)-containing antibodies (BioLegend) for the cell surface markers CD44, CD73, CD90, CD105, and HLA-ABC, and the positive rate of each marker was analyzed using a flow cytometer (Sony).

[0211] Furthermore, umbilical cord-derived mesenchymal stem cells were seeded in 24-well plates (Corning) in the presence of laminin fragments and cultured for one week. Subsequently, the mesenchymal stem cells that adhered to the 24-well plates were immunostained with PE-tagged antibodies for the cell surface markers CD73, CD90, CD105, and HLA-ABC, and bright-field and fluorescence imaging were performed using a fluorescence microscope (Keyence).

[0212] Experiment 4-2 (Comparative Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) at a cell count of 5 x 10 e5 (seeding density 1000 cells / cm2) and seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.) without the addition of iMatrix-511 laminin fragment. Culture was performed using the same procedure as in Experiment 4-1, except that the laminin fragment was not added.

[0213] After culturing, the positivity rates of cell surface markers CD44, CD73, CD90, CD105, and HLA-ABC were analyzed using the same procedure as in Experiment 4-1. Furthermore, the expression of cell surface markers CD73, CD90, CD105, and HLA-ABC was confirmed by immunohistochemical staining, also using the same procedure as in Experiment 4-1.

[0214] (2) Results Figure 13 shows the positivity rates of cell surface markers for umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragments, Figure 14 shows the positivity rates of cell surface markers for adipose-derived mesenchymal stem cells cultured in the presence of laminin fragments, and Figure 15 shows the positivity rates of cell surface markers for umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragments.

[0215] When umbilical cord-derived and adipose-derived mesenchymal stem cells were cultured in the presence of laminin fragments, the positive rates for CD105 and HLA-ABC decreased significantly, but the positive rates for markers other than CD105 and HLA-ABC did not decrease. On the other hand, when umbilical cord-derived mesenchymal stem cells were cultured in the absence of laminin fragments, no significant decrease in the positive rates for CD105 and HLA-ABC was observed.

[0216] When umbilical cord-derived mesenchymal stem cells were cultured in the presence of laminin fragments, immunohistochemical staining images, similar to flow cytometry results, showed positive results for CD73 and CD90 in almost all cells, but very few CD105-positive or HLA-ABC-positive cells were observed. Since the immunohistochemical staining images were taken without proteolytic enzyme treatment of the mesenchymal stem cells, this result demonstrates that the proteolytic enzyme treatment performed for flow cytometry analysis did not reduce the expression of CD105 or HLA-ABC. [Examples]

[0217] Supernatant of mesenchymal stem cell culture In Example 5, mesenchymal stem cells were cultured in proliferation according to the method described in Example 3, followed by multiple production cultures. The culture supernatant was collected after each production culture. The amounts of cytokines, extracellular matrix, and exosome markers contained in the obtained mesenchymal stem cell culture supernatant were analyzed.

[0218] (1) Method The amounts of cytokines and extracellular matrix contained in the culture supernatant were analyzed using an ELISA kit (R&D Systems). Twenty-four cytokines were analyzed: HGF (hepatocyte growth factor), MCP-1, GRO / CXCL1, PDGF-AA, VEGF (vascular endothelial growth factor), TGF-1β, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-1α, IL-1β, TNF-α, IL-8, EOTAXIN, IL-6, G-CSF, GM-CSF, MCP-3, IL-12P40, IP-10, and MIP-1α. Fibronectin was analyzed as an extracellular matrix component. Furthermore, the amount of exosomes in the culture supernatant was analyzed using a CD9 / CD63 ELISA kit (CosmoBio), with exosome marker proteins (CD9 / CD63 fusion proteins) as indicators. In this specification, cytokine levels, extracellular matrix levels, and exosome marker protein levels refer to values ​​measured by ELISA using specific antibodies.

[0219] (2) Results In this example, as in Example 3, the present invention example was cultured in the presence of laminin fragments, while the comparative example was cultured in the absence of laminin fragments. As a result, in the present invention example, multiple production cultures could be repeated, but in the comparative example, cells began to detach from the culture vessel during the first production culture, so only the first production culture could be performed.

[0220] The results for the amount of cytokines contained in the culture supernatant are shown in Figures 16-19 and 21-41, the results for the amount of fibronectin contained in the culture supernatant are shown in Figure 20, and the results for the amount of exosome marker proteins contained in the culture supernatant are shown in Figure 42.

[0221] In Figures 16-42, "1st time" indicates the amount of cytokines, fibronectin, or exosome marker proteins in the culture supernatant collected after the first production culture (4 days) in the example of the present invention, "2nd time" indicates the amount of cytokines, fibronectin, or exosome marker proteins in the culture supernatant collected after the second production culture (4 days) in the example of the present invention, and "3rd time," "4th time," and "5th time" have the same meaning. Also in Figures 16-42, "Comparative Example" indicates the amount of cytokines, fibronectin, or exosome marker proteins in the culture supernatant collected 2 days after the start of the first production culture in the comparative example.

[0222] The results shown in Figures 16-42 indicate that repeated production culture and repeated collection of the culture supernatant according to the method of the present invention tended to increase the amounts of proteins other than IL-1α, IL-1β, and TNF-α (i.e., HGF, MCP-1, GRO / CXCL1, PDGF-AA, VEGF, TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, EOTAXIN, IL-6, G-CSF, GM-CSF, MCP-3, IL-12P40, IP-10, MIP-1α, Fibronectin, and exosome marker proteins). Therefore, it was demonstrated that repeated production culture and repeated collection of the culture supernatant according to the method of the present invention can yield a culture supernatant containing a large amount of cytokines, a large amount of extracellular matrix, and a large amount of exosomes.

[0223] As demonstrated in this example, when mesenchymal stem cell culture supernatant is obtained by culturing in a protein-free culture medium that does not contain foreign components, all the proteins contained in this culture supernatant are derived from mesenchymal stem cells, which has the advantage of being easily applied as a therapeutic agent. Moreover, by repeatedly collecting the mesenchymal stem cell culture supernatant, the content of cytokines such as HGF, fibronectin, and exosomes can be increased, making it superior in that therapeutic effects from these proteins and exosomes can be expected. Furthermore, the content of IL-1α, IL-1β, and TNF-α, which are known to cause inflammation, does not increase when the culture supernatant is repeatedly collected, making it superior in that it has a relatively low content of inflammatory cytokines. [Examples]

[0224] Therapeutic drugs In Example 6, mesenchymal stem cells obtained according to the method of the present invention were administered to rats in a lower limb ischemia model, and the therapeutic effect was confirmed.

[0225] (1) Method <Preparation of rats for a lower limb ischemia model> Twelve-week-old male Sprague Dawley (SD) rats (purchased from CLEA Japan, Osaka, Japan) were used. The SD rats were anesthetized by intraperitoneal administration of a combination anesthetic (M / M / B: 0.3 / 4 / 5) at a dose of 5 ml / kg. Lower limb ischemia was induced by exposing and ligating the external iliac artery and internal iliac vein, the saphenous artery and vein at the level just below the ankle, and all its branches, and then resecting all of the ligated vessels.

[0226] Three days after lower limb ischemia induction, blood flow was measured using laser Doppler perfusion imaging (LDPI). To ensure successful induction of lower limb ischemia and to evaluate the actual effect of cell therapy by excluding rats with significant regenerative capacity, only rats with a relative value (%) of ischemic lower limb blood flow to non-ischemic lower limb blood flow of 60% or less were enrolled for treatment. The enrolled rats were randomly assigned to one of three groups: umbilical cord-derived mesenchymal stem cell (UCMSC) administration, adipose-derived mesenchymal stem cell (ADMSC) administration, or DMEM / F12 administration.

[0227] <Preparation and administration of mesenchymal stem cells> Umbilical cord-derived mesenchymal stem cells (UCMSCs) were cultured in the same manner as in Example 4, both in the presence and absence of laminin fragment. Adipose-derived mesenchymal stem cells (ADMSCs) were also cultured in the same manner as in Example 4, both in the presence and absence of laminin fragment. The resulting mesenchymal stem cells (i.e., umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragment, umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragment, adipose-derived mesenchymal stem cells cultured in the presence of laminin fragment, and adipose-derived mesenchymal stem cells cultured in the absence of laminin fragment) were suspended in 1 ml of DMEM / F12 medium (Sigma) at a cell count of 1.25 x 10 e6.

[0228] The cell suspension was administered intravenously via the tail vein using a 27-gauge needle. Administration was performed four times in total, on days 4, 5, 6, and 10, after induction of lower limb ischemia. DMEM / F12 medium alone was administered as a control.

[0229] <Measuring blood flow> Lower extremity blood flow was evaluated using the Moor LDI 2.0 system (Moor Instruments, Devon, UK). Blood flow was measured 14 days after induction of lower extremity ischemia. Rats were anesthetized by isoflurane inhalation at the time of blood flow measurement. The blood flow ratio (%) was calculated as a relative value using the following formula. Blood flow ratio (%) = (Blood flow in the left ischemic lower limb / Blood flow in the right non-ischemic lower limb) × 100

[0230] (2) Results The results for the lower extremity blood flow ratio are shown in Figure 43. In Figure 43, "Control" represents the group administered DMEM / F12 medium, "UCMSC laminin-" represents the group administered umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragment, "UCMSC laminin+" represents the group administered umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragment, "ADMSC laminin-" represents the group administered adipose-derived mesenchymal stem cells cultured in the absence of laminin fragment, and "ADMSC laminin+" represents the group administered adipose-derived mesenchymal stem cells cultured in the presence of laminin fragment. In Figure 43, the error bars represent the standard error.

[0231] "UCMSC laminin+" and "ADMSC laminin+" showed a significantly increased blood flow ratio compared to the control group, and a statistically significant difference was confirmed by a t-test. On the other hand, While "UCMSC laminin-" and "ADMSC laminin-" showed a slight tendency towards increased blood flow ratio compared to the control, no significant difference was observed compared to the control by t-test.

[0232] These results indicate that mesenchymal stem cells obtained according to the method of the present invention are effective as a therapeutic agent for lower limb ischemia. Since this therapeutic effect is thought to be brought about by cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells, it is believed that the culture supernatant of mesenchymal stem cells obtained according to the method of the present invention also has a similar therapeutic effect. Furthermore, since this therapeutic effect is thought to be brought about by cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells, it is believed that mesenchymal stem cells and their culture supernatant obtained according to the method of the present invention also have therapeutic effects against diseases in which cytokines are known to exert therapeutic effects, diseases in which extracellular matrix is ​​known to exert therapeutic effects, and diseases in which exosomes are known to exert therapeutic effects.

Claims

1. A method for producing a culture supernatant of mesenchymal stem cells, Mesenchymal stem cells are cultured in a growth culture medium in the presence of a culture substrate selected from laminin fragments having integrin-binding activity and their variants, by adherent culture, to obtain proliferating cells in an adherent state. The proliferating cells are cultured in a production culture medium while maintaining the adhesion state, and the cells are made to produce cell products. The process includes culturing the proliferating cells in the production culture medium, while maintaining the adherent state, after the culture in the production culture medium, The cultivation in the production culture medium and the cultivation in the recovery culture medium are repeated alternately while maintaining the adhesion state of the cells, and the method further includes collecting the supernatant of the production culture medium after the cultivation in the production culture medium. The aforementioned proliferation culture medium is a culture medium used to proliferate mesenchymal stem cells and obtain proliferating cells in an adherent state. The aforementioned production culture medium is a culture medium used to induce adhering proliferating cells to produce cell products and to collect the supernatant. The method comprises a recovery culture medium being a culture medium used to provide cells that have been cultured for production to the next production culture while maintaining their adherence.

2. The method according to claim 1, wherein the growth medium, the production medium, and the recovery medium are serum-free.

3. The method according to claim 1 or 2, wherein the growth medium, the production medium, and the recovery medium are xenofree.

4. The method according to claim 1, wherein the collection of the supernatant of the production culture medium is performed after the second and subsequent culturing in the production culture medium.

5. The method according to any one of claims 1 to 4, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

6. The method according to any one of claims 1 to 5, wherein the culture supernatant of the mesenchymal stem cells contains 5000 pg / mL or more of HGF.

7. The method according to any one of claims 1 to 5, wherein the culture supernatant of the mesenchymal stem cells contains 10,000 pg / mL or more of HGF.

8. The method according to any one of claims 1 to 5, wherein the culture supernatant of the mesenchymal stem cells contains 50 pg / mL or more of CD9 / CD63EC domain fusion protein.

9. The method according to any one of claims 1 to 5, wherein the culture supernatant of the mesenchymal stem cells contains 100 pg / mL or more of CD9 / CD63EC domain fusion protein.

10. The method according to any one of claims 1 to 5, wherein the culture supernatant of the mesenchymal stem cells contains 200 pg / mL or more of CD9 / CD63EC domain fusion protein.

11. The method according to any one of claims 6 to 10, wherein the culture supernatant of the mesenchymal stem cells further contains 3000 pg / mL or more of MCP-1, 1000 pg / mL or more of GRO, and 5 μg / mL or more of fibronectin.

12. The method according to any one of claims 6 to 10, wherein the culture supernatant of the mesenchymal stem cells further contains 200 pg / mL or more of TGF-1b, 5 pg / mL or more of IL-4, and 8 pg / mL or more of IL-10.

13. The method according to any one of claims 6 to 10, wherein the culture supernatant of the mesenchymal stem cells does not contain at least one of insulin, transferrin, and albumin.

14. The method according to any one of claims 6 to 10, wherein the culture supernatant of the mesenchymal stem cells does not contain recombinant protein.

15. The method according to any one of claims 6 to 10, wherein the amount of at least one of IL-1α, IL-1β, and TNF-α contained in the culture supernatant of the mesenchymal stem cells is 0 to 15 pg / mL.

16. The method according to any one of claims 6 to 10, wherein the culture supernatant of the mesenchymal stem cells contains 20 or more types of cytokines, and each cytokine has a concentration of 10 pg / mL or more.

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