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

Abstract

A method for producing proliferating cells, said method comprising adhesion-culturing cells, which have been sown at a cell density of 0.002-2000 cells / cm2, in a proliferation culture solution in the presence of a culture substrate selected from a laminin fragment having integrin binding activity and a variant thereof and thus proliferating the cells.

Description

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

[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.

[0002] Stem cells, such as mesenchymal stem cells, are attracting worldwide attention for their potential application in regenerative medicine. To apply stem cells to regenerative medicine, it is necessary to develop culture techniques that enable stable cultivation and proliferation of these cells.

[0003] In order to culture, proliferate, and mass-produce stem cells, a common procedure is to first seed the cells into a culture vessel at a certain cell density and culture them until 80 to 90 percent or more of the surface area of ​​the culture vessel is covered with cells (a so-called confluent state), after which the cells are detached with an enzyme such as trypsin, and the resulting cells are passaged into multiple 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 various cell-derived components such as cytokines and exosomes, and are expected to be applied in medical treatments.

[0005] International Publication No. 2014 / 035215

[0006] When using cell culture technology to mass-produce proliferated cells and cell-derived components, it is necessary to efficiently proliferate the cells and efficiently recover the culture supernatant containing the cell-derived components.

[0007] Therefore, an object of the present invention is to provide a cell culture technique that allows cells seeded at a low cell density to grow at a high growth rate. Another object of the present invention is to provide a cell culture technique that is a further development of the above-mentioned cell culture technique, which allows for the production of large amounts of cell products in a simple manner while maintaining the adherent state of the resulting proliferated cells. Furthermore, an object of the present invention is to provide a mesenchymal stem cell population with novel characteristics based on the above-mentioned cell culture technique, and to provide a stem cell culture supernatant containing large amounts of cell products such as cytokines based on the above-mentioned cell culture technique. Another object of the present invention is to provide a therapeutic agent containing the above-mentioned mesenchymal stem cell population or the above-mentioned culture supernatant.

[0008] In one aspect, 0.002 to 2000 cells / cm 2 and culturing the cells in a growth medium by adhesion culture in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin binding activity, thereby growing the cells.

[0009] According to another aspect, there is provided a method for producing a cell product, comprising: culturing cells in a growth culture medium according to the above-described "method for producing proliferating cells" to obtain proliferating cells in an adherent state; and culturing the proliferating cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product. According to yet another aspect, there is provided a method for producing a cell product, comprising: culturing cells in a growth culture medium according to the above-described "method for producing proliferating cells" to obtain proliferating cells in an adherent state; culturing the proliferating cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product; and, after the culturing in the production culture medium, culturing the proliferating cells in a recovery culture medium while maintaining the adherent state, wherein the culturing in the production culture medium and the culturing in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells.

[0010] According to yet another aspect, there is provided a method for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, which comprises culturing mesenchymal stem cells in a growth medium in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin-binding activity to obtain proliferated cells. According to yet another aspect, there is provided a mesenchymal stem cell population in which the rate of HLA-ABC-positive mesenchymal stem cells is 70% or less. According to yet another aspect, there is provided a method for producing a stem cell culture supernatant, comprising: culturing stem cells in a growth culture medium by adhesion culture in the presence of a culture substrate selected from a laminin fragment and its variant having integrin binding activity to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product; and, after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells, and the method further comprises recovering the supernatant of the production culture medium after the culture in the production culture medium. According to yet another aspect, there is provided a stem cell culture supernatant containing 5000 pg / mL or more of HGF. According to yet another aspect, there is provided a stem cell culture supernatant containing 50 pg / mL or more of CD9 / CD63 EC domain fusion protein. In yet another aspect, there is provided a therapeutic agent comprising the above-mentioned mesenchymal stem cell population or the above-mentioned culture supernatant.

[0011] According to the present invention, a cell culture technique can be provided that allows cells seeded at a low cell density to grow at a high growth rate. Furthermore, according to the present invention, a cell culture technique that is a further development of the above-mentioned cell culture technique can be provided that allows for the production of large amounts of cell products by a simple method while maintaining the adherent state of the obtained proliferated cells. Based on the above-mentioned cell culture technique, the present invention can provide a mesenchymal stem cell population with novel characteristics, and a stem cell culture supernatant containing large amounts of cell products such as cytokines. Furthermore, according to the present invention, a therapeutic agent containing the above-mentioned mesenchymal stem cell population or the above-mentioned culture supernatant can be provided.

[0012] Graph showing the number of proliferating cells (Comparative Example) Seeding density 10 cells / cm 2 Microscopic images of cells after 20 days of culture at a seeding density of 10 cells / cm (Comparative Example). Graph showing the number of proliferating cells (Example of the present invention). 2 Microscopic image of cells after 20 days of culture at a seeding density of 10 cells / cm (Example of the present invention). Graph showing the number of proliferating cells (Reference Example). 23. A microscopic image of cells after 20 days of culture in 100% ethanol at 20°C (Reference Example). A microscopic image of cells after 3 days of production culture (Comparative Example). A microscopic image of cells after 3 days of production culture (Comparative Example). A microscopic image of cells after 3 days of production culture (Example of the present invention). A microscopic image of cells after 3 days of production culture (Example of the present invention). A schematic diagram of the culture steps performed in Example 3. A graph showing the results of cytokine quantification. A graph showing the results of cytokine quantification. A graph showing the results of cytokine quantification. A graph showing the positive rate of cell surface markers in umbilical cord-derived mesenchymal stem cells when cultured in the presence of laminin fragments. A graph showing the positive rate of cell surface markers in adipose-derived mesenchymal stem cells when cultured in the presence of laminin fragments. A graph showing the positive rate of cell surface markers in umbilical cord-derived mesenchymal stem cells when cultured in the absence of laminin fragments. A graph showing the amount of HGF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. A graph showing the amount of HGF contained in the culture supernatant of adipose-derived mesenchymal stem cells. Graph showing the amount of MCP-1 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of GRO / CXCL1 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of fibronectin contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of PDGF-AA contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of VEGF contained in the culture supernatant of adipose-derived mesenchymal stem cells. Graph showing the amount of TGF-1b contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-4 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-10 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-13 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-7 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-15 contained in the culture supernatant of adipose-derived mesenchymal stem cells. Graph showing the amount of IL-9 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-1α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-1β contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of TNF-α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-8 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of EOTAXIN contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells.Graph showing the amount of IL-6 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of G-CSF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of GM-CSF contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of MCP-3 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IL-12P40 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of IP-10 contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of MIP-1α contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing the amount of exosome marker protein contained in the culture supernatant of umbilical cord-derived mesenchymal stem cells. Graph showing lower limb blood flow ratio.

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

[0014] When cells are propagated by subculture, the lower the cell density at seeding, the more culture vessels will be cultured in the next generation from 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, typical split ratios for stem cells are around 1:5 to 1:20, and stem cells are rarely seeded at low cell densities such as 1:100 or 1:1000. In practice, stem cells can be seeded at a density of 2,000 cells / cm. 2 The cells were seeded at a density exceeding 1000 and passaged.

[0015] Meanwhile, research is being conducted into the cultivation of 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 the treatment of diseases. In this case, it is undesirable to culture stem cells in a culture medium containing animal-derived components, human serum, or exogenous cytokines or recombinant proteins such as insulin, and then purify and use the resulting supernatant components. This is because there is a risk that the above-mentioned exogenous components other than the 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 exogenous components for treatment.

[0016] Under such technical background, the present inventors have developed a method for culturing stem cells at a density of 10,000 cells / cm. 2 When the cells were seeded and cultured at a cell density of 1000 cells / cm, the cells could be grown to a confluent state, but when the stem cells were seeded at a cell density of 1000 cells / cm, the cells could be grown to a confluent state. 2 When the cells were seeded and cultured at a cell density lower than 1000 cells / cm, cell proliferation stopped and they could not be grown to a confluent state (see Example 1, Figures 1 and 2 below). 2 The cells were seeded at a cell density of 1000 and cultured, and the resulting proliferated cells were cultured in a protein-free culture medium that did not contain any exogenous components (i.e., production culture was performed). However, during the production culture, the cells detached from the culture vessel, and the adherent state of the proliferated cells could not be maintained (see Example 2, FIGS. 7A and 7B, described below).

[0017] The present inventors have attempted to solve these problems. As a result, the present inventors have found that, first, 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 laminin fragments (hereinafter simply referred to as laminin fragments) having integrin-binding activity (see Example 1, Figures 3 and 4, described below). Second, the present inventors have found that when stem cells are cultured and proliferated in the presence of laminin fragments, the resulting proliferated cells do not detach from the culture vessel during the culture process, even when cultured in a protein-free medium containing no exogenous components, and the proliferated cells can maintain an adherent state (see Example 2, Figures 8A and 8B, described below). Third, the inventors cultured the above-mentioned proliferating cells in a protein-free culture medium that does not contain any exogenous components (i.e., performed production culture), and then further cultured them in a cell proliferation culture medium (i.e., performed recovery culture), and then performed production culture again.They found that production culture could be repeated while maintaining the adherent state of the proliferating cells, and that cell-derived components could be produced over a long period of time (see Example 3 below).

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

[0019] 1. Method for Producing Proliferating Cells According to one aspect, the method for producing proliferating cells is a method for producing proliferating cells at a density of 0.002 to 2000 cells / cm. 2 and culturing the cells in a growth medium by adhesion culture in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin binding activity, thereby growing the cells.

[0020] Preferably, the culture can be carried out in a growth medium containing the culture substrate (i.e., laminin fragment or its variant having integrin binding activity). That is, according to a preferred embodiment, the culture can be carried out at a density of 0.002 to 2000 cells / cm. 2and culturing the cells seeded at a cell density of 1000 to 15000 by adhesion culture in a growth medium containing a laminin fragment (hereinafter simply referred to as laminin fragment) having integrin binding activity or a modified form thereof, thereby growing the cells.

[0021] According to a specific example, the method for producing proliferated cells comprises preparing a growth culture medium containing a laminin fragment or a variant thereof in advance, and culturing the cells at a density of 0.002 to 2000 cells / cm. 2 and seeding the resulting cell suspension into a culture vessel; and culturing the cells in the growth culture medium by adhesion culture to grow the cells.

[0022] Alternatively, according to another specific example, the method for producing expanded cells comprises: culturing the cells at a density of 0.002 to 2000 cells / cm 2 the laminin fragment or its variant is added to the obtained cell suspension, and the resultant is seeded in a culture vessel; and the cells are cultured in the growth culture medium by adhesion culture to grow the cells.

[0023] Alternatively, according to another specific example, the method for producing expanded cells comprises: culturing the cells at a density of 0.002 to 2000 cells / cm 2 the cells in a growth medium to a seeding density of 1000 μg / ml, seeding the obtained cell suspension in a culture vessel, and adding a laminin fragment or a variant thereof to the seeded cell suspension; and culturing the cells in the growth medium by adhesion culture to grow the cells.

[0024] (Cells) Any cells, for example, stem cells, can be used as the cells. The cells are preferably mesenchymal stem cells, induced pluripotent stem cells (iPS cells), or embryonic stem cells (ES cells), more preferably mesenchymal stem cells. The cells are more preferably 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. The cells are preferably human mesenchymal stem cells, human induced pluripotent stem cells (iPS cells), or human embryonic stem cells (ES cells), more preferably human mesenchymal stem cells. The cells are more preferably 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, 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 expanded cells or cell-derived components, the expanded cells or cell-derived components can be produced as needed by using cryopreserved cells as starting cells and expanding them according to the method of the present invention.

[0027] (Seeding) In this method, cells are seeded at 0.002 to 2000 cells / cm. 2 The cells are seeded in growth medium at a cell density of 1000 kJ / well.

[0028] The cells are preferably 0.002 to 1900 cells / cm 2 , more preferably 0.002 to 1800 cells / cm 2 , more preferably 0.002 to 1700 cells / cm 2 , more preferably 0.002 to 1600 cells / cm 2 , more preferably 0.002 to 1500 cells / cm 2 , more preferably 0.002 to 1400 cells / cm 2 , more preferably 0.002 to 1300 cells / cm 2, more preferably 0.002 to 1200 cells / cm 2 , more preferably 0.002 to 1100 cells / cm 2 , more preferably 0.002 to 1000 cells / cm 2 , more preferably 0.002 to 900 cells / cm 2 , more preferably 0.002 to 800 cells / cm 2 , more preferably 0.002 to 700 cells / cm 2 , more preferably 0.002 to 600 cells / cm 2 , more preferably 0.002 to 500 cells / cm 2 , more preferably 0.002 to 400 cells / cm 2 , more preferably 0.002 to 300 cells / cm 2 , more preferably 0.002 to 200 cells / cm 2 , more preferably 0.002 to 100 cells / cm 2 The cells are seeded at a cell density of 1000 x g.

[0029] Considering the time it takes for the cells to reach confluence, the cells are preferably cultured at a density of 1 to 2000 cells / cm. 2 , more preferably 1 to 1900 cells / cm 2 , more preferably 1 to 1800 cells / cm 2 , more preferably 1 to 1700 cells / cm 2 , more preferably 1 to 1600 cells / cm 2 , more preferably 1 to 1500 cells / cm 2 , more preferably 1 to 1400 cells / cm 2 , more preferably 1 to 1300 cells / cm 2 , more preferably 1 to 1200 cells / cm 2 , more preferably 1 to 1100 cells / cm 2 , more preferably 1 to 1000 cells / cm 2 , more preferably 1 to 900 cells / cm 2 , more preferably 1 to 800 cells / cm 2 , more preferably 1 to 700 cells / cm 2 , more preferably 1 to 600 cells / cm 2 , more preferably 1 to 500 cells / cm 2, more preferably 1 to 400 cells / cm 2 , more preferably 1 to 300 cells / cm 2 , more preferably 1 to 200 cells / cm 2 , more preferably 1 to 100 cells / cm 2 The cells are seeded at a cell density of 1000 x g.

[0030] The above-mentioned cell density is lower than the seeding density typically used for stem cell expansion culture. Cells are typically seeded in a single-cell state. Single-cell states can be prepared by treating cell clusters with proteolytic enzymes (e.g., trypsin).

[0031] The growth medium may be any known cell growth medium depending on the type of cells. For example, in the case of human stem cells, a commercially available growth medium for human stem cells may 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 growth medium be serum-free and xeno-free.

[0033] Furthermore, whereas the "production culture medium" described below preferably does not contain proteins, the proliferation culture medium preferably contains proteins that promote cell proliferation. Examples of proteins that promote stem cell proliferation include basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGFβ1), epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin, albumin, and transferrin. More specifically, the proliferation culture medium may be a medium prepared by adding growth factors to a basal 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 that can be used include, for example, 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). All of these growth culture media are serum-free and xeno-free.

[0035] Any vessel used for cell adhesion culture can be used as the culture vessel. Generally, flat-bottom vessels such as culture flasks, culture dishes, and culture plates can be used. The use of a culture vessel with a large bottom area allows the production of a large amount of proliferated cells and cell-derived components from a single culture vessel. For example, a 500 cm 2 or more, preferably 500 to 10,000 cm 2 It is desirable to use a culture vessel having a base area of ​​1000 μm or more.

[0036] (Expansion culture) 0.002 to 2000 cells / cm 2 The cells seeded at a cell density of 1000 μg / ml are cultured in a growth medium by adhesion culture in the presence of a laminin fragment or a variant thereof having 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 referred to simply as a laminin fragment) or a modified form thereof," the stem cells can be grown 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 culture the cells until they reach a confluent state. As used herein, "confluent state" refers to a state in which the cells cover 80% or more of the bottom area of ​​the culture vessel. More preferably, the culture can be carried out until the cells cover 80 to 90% of the bottom area of ​​the culture vessel.

[0039] Culturing in the presence of a laminin fragment or its variant may be performed using a growth medium containing the laminin fragment or its variant, or using a culture vessel pre-coated with the laminin fragment or its variant. Culturing using a growth medium containing the laminin fragment or its variant is preferred because it requires less laminin fragment or its variant and is easier to handle than culturing using a culture vessel pre-coated with the laminin fragment or its variant.

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

[0041] The laminin fragment may be a laminin fragment known to have integrin-binding activity. It has been reported that pre-coating a culture vessel with a "laminin fragment having integrin-binding activity" enables human ES cells and human iPS cells to 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 widespread because it does not use xenogeneic components derived from animals. Therefore, laminin fragments used in the art for growing human ES cells and human iPS cells in feeder-free culture can also be used in the method of the present invention.

[0042] The laminin fragment is preferably a human-derived laminin fragment, since it is preferable that the growth medium does not contain any xenogeneic components.

[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 this 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 to α5, three types of β chains, β1 to β3, and three types of γ chains, γ1 to 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, and laminin-121 E8 fragment.

[0045] As a modified laminin fragment, a known complex composed of a laminin fragment having integrin-binding activity and another functional molecule can be used, such as 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] A preferred laminin fragment variant is a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule. The laminin fragment contained in such a complex may be any of the laminin fragments 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] The laminin fragment variant is more preferably a complex of a laminin E8 fragment and a growth factor-binding molecule. The laminin E8 fragment contained in such a complex is preferably the laminin E8 fragment exemplified above. The growth factor-binding molecule contained in such a complex is preferably heparan sulfate. Therefore, the laminin fragment variant is even more preferably a complex of the laminin E8 fragment exemplified above and heparan sulfate. The laminin fragment variant is most preferably a complex of the laminin 511 E8 fragment and heparan sulfate or a complex of the laminin 421 E8 fragment and heparan sulfate.

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

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

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

[0051] As mentioned above, the culture can be carried out in any culture vessel, for example, a 500 cm 2 or more, preferably 500 to 10,000 cm 2 During the culture, the growth medium may be appropriately replaced with a fresh one having the same composition.

[0052] (Effects) Conventionally, when cells are seeded at a low cell density, cell growth stops and they cannot be grown to a confluent state, whereas the above-described method allows cells seeded at a low cell density to grow at a high growth rate. This makes it possible to increase the number of culture vessels in which cells from one culture vessel are cultured in the next generation, and enables efficient production of proliferated cells and cell-derived components.

[0053] 2. Cell Product Production Method 2-1. First Embodiment (Embodiment in which a Single Production Culture Step is Performed) According to another aspect, there is provided a cell product production method, comprising: culturing cells in a growth culture medium according to the above-described "production method of proliferating cells" to obtain proliferating cells in an adherent state; and culturing the proliferating cells in a production culture medium while maintaining the adherent state, to cause the cells to produce a cell product.

[0054] That is, the method for producing the cell product is 0.002 to 2000 cells / cm 2 culturing the cells seeded at a cell density of 0.1% by adhesion culture in a growth culture medium in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin binding activity to obtain proliferated cells in an adherent state; and culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product.

[0055] In the following description, culture in a growth medium is referred to as "growth culture," and culture in a production medium is referred to as "production culture."

[0056] As described above, the growth culture is preferably carried out in a growth culture medium containing a laminin fragment or a variant thereof having integrin binding activity. That is, in a preferred embodiment, the growth culture can be carried out at a density of 0.002 to 2000 cells / cm. 2 in a growth culture medium containing a laminin fragment or a variant thereof having integrin binding activity to obtain proliferated cells in an adherent state; and culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product.

[0057] (Expansion culture) The steps up to expansion culture can be carried out as explained in the section <1. Method for producing expanded cells>. This allows expanded cells to be obtained in an adherent state. If expansion culture is carried out until the cells reach a confluent state, expanded cells can be obtained in a confluent state.

[0058] (Production Culture) After growth culture, the proliferating cells are cultured in a production culture medium while maintaining an adherent state. "Culturing the proliferating cells in a production culture medium while maintaining an adherent state" means that, after growth culture, the cells that have adhered to the bottom of the culture vessel are not detached from the bottom of the culture vessel, and production culture is performed while maintaining the adherent state.

[0059] Production culture can be carried out by replacing the growth culture medium in the culture vessel with a production culture medium after growth culture, which allows the grown cells to be cultured in the production culture medium while maintaining their adherent state.

[0060] During the production culture, cells can produce cell products and release them into the production culture medium. Cell products are any substances released by cells into the production culture medium, including, for example, cell metabolites such as amino acids, lipids, and sugars; hormones; peptides; secreted proteins such as cytokines and extracellular matrix; and exosomes. For example, stem cells can produce various cytokines and exosomes and release them into the production culture medium. Therefore, the method for producing a cell product may further include a step of recovering the supernatant of the production culture medium after the production culture.

[0061] The supernatant of the production culture obtained in the recovery step is expected to be used for therapeutic purposes such as disease treatment and regenerative medicine. Therefore, it is desirable that the production culture medium is a culture medium that does not contain xenogeneic components. When the cells being cultured are human cells, xenogeneic components refer to components derived from animals other than humans. It is also desirable that the production culture medium is a culture medium that does not contain cytokines or insulin. It is also desirable that the production culture medium is a culture medium that does not contain proteins. It is also desirable that the production culture medium is a culture medium that does not contain serum.

[0062] More preferably, the production culture medium is a cell culture medium that is free of xenogeneic components and is free of cytokines and insulin. Even more preferably, the production culture medium is a cell culture medium that is free of xenogeneic components, cytokines and insulin, and is free of proteins. Even more preferably, the production culture medium is a cell culture medium that is free of xenogeneic components, cytokines and insulin, is free of proteins, and is free of serum.

[0063] The production culture medium can be a cell culture medium appropriate for the cell type that does not contain the above-mentioned components (i.e., xenogeneic components, cytokines, insulin, proteins, and human serum). More specifically, the production culture medium can be a basal cell culture medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI 1640, etc.), or a basal cell culture medium supplemented with cell nutrients. Basal cell culture media are commercially available and generally contain amino acids, vitamins, inorganic salts, and a carbon source. The production culture medium does not need to contain laminin fragments or their modifications.

[0064] For human mesenchymal stem cells, the production culture medium can be, for example, DMEM / F12 medium supplemented with amino acids. The added amino acids can be commercially available amino acid solutions for medium supplementation, such as MEM essential amino acid solution (Fujifilm Wako Pure Chemical Corporation) or MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Corporation). This production culture medium does not contain any xenogeneic components, cytokines, insulin, proteins, or human serum.

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

[0066] (Effects) Conventionally, cell growth has stopped during expansion culture, or cells have detached from the culture vessel during production culture, making it impossible to maintain the cell adhesion state. However, according to the above-mentioned method, cells seeded at a low cell density can be expanded at a high expansion rate, and then production culture can be performed while maintaining the adhesion state of the expanded cells. This allows for the production of large amounts of cell products using a simple method.

[0067] Specifically, the above-described method allows cells seeded at a low cell density to grow at a high growth rate, thereby enabling the production of a large amount of cell product from a small amount of starting cells. Furthermore, the above-described method is convenient in that it maintains the adherent state of the cells, allowing the transition from expansion culture to production culture simply by changing the culture medium. In other words, the above-described method does not require cell passaging, thereby eliminating lot-to-lot variations and the costs associated with passaging. Furthermore, the above-described method is convenient in that it maintains the adherent state of the cells, allowing the culture supernatant containing the cell product to be easily recovered.

[0068] 2-2. Second embodiment (embodiment in which production culture steps are performed multiple times) In another aspect, there is provided a method for producing a cell product, comprising: culturing cells in a growth culture medium according to the above-described "method for producing proliferating cells" to obtain proliferating cells in an adherent state; culturing the proliferating cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product; and, after the culture in the production culture medium, culturing the proliferating cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells.

[0069] That is, the method for producing the cell product is 0.002 to 2000 cells / cm 2 in a growth culture medium in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity, to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state, to cause the cells to produce a cell product; and after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells.

[0070] In the following description, culture in a growth medium is referred to as "growth culture," culture in a production medium is referred to as "production culture," and culture in a recovery medium is referred to as "recovery culture."

[0071] As described above, the growth culture is preferably carried out in a growth culture medium containing a laminin fragment or a variant thereof having integrin binding activity. That is, in a preferred embodiment, the growth culture can be carried out at a density of 0.002 to 2000 cells / cm. 2 in a growth culture medium containing a laminin fragment or a variant thereof having integrin-binding activity to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product; and after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells.

[0072] (Expansion culture) The steps up to expansion culture can be carried out as explained in the section <1. Method for producing expanded cells>. This allows expanded cells to be obtained in an adherent state. If expansion culture is carried out until the cells reach a confluent state, expanded cells can be obtained in a confluent state.

[0073] (Production culture) The production culture step can be carried out as described in the section <2-1. First embodiment (embodiment in which one production culture step is carried out)>. This allows the cells to produce a cell product.

[0074] (Recovery Culture) After the production culture, the proliferated cells are cultured in a recovery culture medium while maintaining their adherent state. "Culturing the proliferated cells in a recovery culture medium while maintaining their adherent state" means that, after the production culture, recovery culture is performed on the cells that have adhered to the bottom of the culture vessel without detaching them from the bottom of the culture vessel while maintaining their adherent state.

[0075] Recovery culture can be performed by replacing the production culture medium in the culture vessel with a recovery culture medium after production culture, allowing the proliferating cells to be cultured in the production culture medium while maintaining an adherent state, and then further cultured in the recovery culture medium while maintaining an adherent state.

[0076] During recovery culture, the cells can be restored to a state where they can again produce a sufficient amount of the cell product. That is, recovery culture is culture carried out to restore the ability of the cells to produce the cell product for the next production culture step.

[0077] Recovery culture can be performed using a culture medium used in cell proliferation culture. That is, a known cell proliferation culture medium can be used as the recovery culture medium depending on the type of cell. For example, in the case of human stem cells, a commercially available medium for proliferation of human stem cells 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 medium be a serum-free and xeno-free medium.

[0079] Furthermore, while the above-mentioned "production culture medium" 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 basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGFβ1), epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin, albumin, and transferrin. More specifically, the recovery culture medium may be a medium prepared by adding growth factors to a basal cell culture medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.).

[0080] The recovery medium may have the same composition as the growth medium, but does not need to contain laminin fragments or their variants.

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

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

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

[0084] (Effects) As with the method according to the first embodiment, the method according to the second embodiment allows cells seeded at a low cell density to grow at a high growth rate, and then allows production culture to be performed while maintaining the adherent state of the resulting grown cells. In addition, the method according to the second embodiment allows recovery culture to be performed while maintaining the adherent state of cells after production culture, thereby restoring the ability of the cells to produce a cell product. This allows the production culture process to be repeated multiple times while maintaining the adherent state of the cells, resulting in the long-term production of large amounts of cell products using a simple method.

[0085] Specifically, the method according to the second embodiment allows cells seeded at a low cell density to grow at a high growth rate, thereby enabling the production of a large amount of cell product from a small amount of starting cells. Furthermore, the method according to the second embodiment is convenient in that it maintains the adherent state of the cells, allowing for the transition from expansion culture to production culture, or for repeating cycles of production culture and recovery culture, simply by changing the culture medium. In other words, the method according to the second embodiment does not require cell passaging, thereby eliminating lot-to-lot variations and the costs associated with passaging. Furthermore, the method according to the second embodiment is convenient in that it maintains the adherent state of the cells, allowing for the easy recovery of the culture supernatant containing the cell product.

[0086] In particular, the method according to the second embodiment has the advantage that a cell product can be produced each time a cycle of production culture and recovery culture is repeated, thereby enabling substance production over a long period of time. Furthermore, the method according to the second embodiment has the advantage that the amount of cell product produced does not decrease even when the cycle of production culture and recovery culture is repeated, thereby enabling continuous production of a large amount of cell product.

[0087] 3. Preferred embodiments The preferred embodiments of the present invention are summarized below. [A1] 0.002 to 2000 cells / cm 2 [A2] A method for producing proliferated cells, comprising culturing cells seeded at a cell density of 0.002 to 2,000 cells / cm by adhesion culture in a growth medium in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity, thereby proliferating the cells. 2 [A3] A method for producing proliferated cells, comprising culturing cells seeded at a cell density of 0.002 to 2,000 cells / cm by adhesion culture in a growth culture medium containing a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity, thereby proliferating the cells. 2[A4] A method for producing proliferated cells, comprising: seeding a cell suspension containing cells in an amount sufficient to achieve a seeding density of 0.002 to 2,000 cells / cm and a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity into a culture vessel; and then culturing the cells in the growth culture medium by adhesion culture to proliferate the cells. 2 A method for producing proliferated cells, comprising: seeding a cell suspension containing cells in an amount sufficient to achieve a seeding density of 1000 to 15000 in a culture vessel, adding a culture substrate selected from a laminin fragment and a modified form thereof having integrin binding activity to the seeded cell suspension, and then culturing the cells in the growth medium by adhesion culture to proliferate the cells. [A5] The method according to any one of [A1] to [A4], wherein the cells are stem cells.

[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 prepared by thawing frozen cells. [A17] The cells are cultured at a density of 0.002 to 1900 cells / cm. 2 , preferably 0.002 to 1800 cells / cm 2 , more preferably 0.002 to 1700 cells / cm 2 , more preferably 0.002 to 1600 cells / cm 2 , more preferably 0.002 to 1500 cells / cm 2 , more preferably 0.002 to 1400 cells / cm 2 , more preferably 0.002 to 1300 cells / cm 2 , more preferably 0.002 to 1200 cells / cm 2 , more preferably 0.002 to 1100 cells / cm 2 , more preferably 0.002 to 1000 cells / cm 2 , more preferably 0.002 to 900 cells / cm 2 , more preferably 0.002 to 800 cells / cm 2 , more preferably 0.002 to 700 cells / cm 2 , more preferably 0.002 to 600 cells / cm 2 , more preferably 0.002 to 500 cells / cm 2, more preferably 0.002 to 400 cells / cm 2 , more preferably 0.002 to 300 cells / cm 2 , more preferably 0.002 to 200 cells / cm 2 , more preferably 0.002 to 100 cells / cm 2 [A18] The method according to any one of [A1] to [A16], wherein the cells are seeded at a cell density of 1 to 2,000 cells / cm. 2 , preferably 1 to 1900 cells / cm 2 , more preferably 1 to 1800 cells / cm 2 , more preferably 1 to 1700 cells / cm 2 , more preferably 1 to 1600 cells / cm 2 , more preferably 1 to 1500 cells / cm 2 , more preferably 1 to 1400 cells / cm 2 , more preferably 1 to 1300 cells / cm 2 , more preferably 1 to 1200 cells / cm 2 , more preferably 1 to 1100 cells / cm 2 , more preferably 1 to 1000 cells / cm 2 , more preferably 1 to 900 cells / cm 2 , more preferably 1 to 800 cells / cm 2 , more preferably 1 to 700 cells / cm 2 , more preferably 1 to 600 cells / cm 2 , more preferably 1 to 500 cells / cm 2 , more preferably 1 to 400 cells / cm 2 , more preferably 1 to 300 cells / cm 2 , more preferably 1 to 200 cells / cm 2 , more preferably 1 to 100 cells / cm 2 [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 growth 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 basal cell culture medium supplemented with a growth factor.

[0091] [A21] The method according to any one of [A1] to [A20], wherein the growth culture medium is a basal medium for cell culture supplemented with growth factors. [A22] The method according to any one of [A1] to [A21], wherein the culture is continued until the cells reach a confluent state. [A23] The method according to any one of [A1] to [A21], wherein the culture is continued until the cells reach a confluent state. 2 [A24] The method according to any one of [A1] to [A22], wherein the culture is carried out in a culture vessel having a bottom area of ​​500 to 10,000 cm or more. 2 [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 a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a 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 that has 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 a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a 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 molecule 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 molecule is a complex of laminin 421 E8 fragment and heparan sulfate. [A40] The concentration of the laminin fragment or its modified molecule in the growth culture medium is 1 / 400 of the total volume of the growth factor binding molecule per 1 cm of culture area of ​​the culture vessel. 2 [A41] 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 cm of culture area of ​​the culture vessel. 2[A42] 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 cm of culture area of ​​the culture vessel. 2 The method according to any one of [A1] to [A41], wherein the amount is 0.05 μg to 0.25 μg per unit area.

[0095] [B1] A method for producing a cell product, comprising: culturing cells in a growth culture medium according to the method of any one of [A1] to [A42] to obtain proliferated cells in an adherent state, and culturing the proliferated cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product. [B2] A method for producing a cell product, comprising: culturing cells in a growth culture medium according to the method of any one of [A1] to [A42] to obtain proliferated cells in an adherent state, culturing the proliferated cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product, and after the culturing in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culturing in the production culture medium and the culturing in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells. [B3] The method according to [B1] or [B2], wherein the cell product is a cell metabolite such as an amino acid, lipid, or sugar; a hormone; a peptide; a secreted protein such as a cytokine or extracellular matrix; or an exosome. [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 containing no xenogenous components. [B7] The method according to any one of [B1] to [B6], wherein the production culture medium is a culture medium containing no cytokines or insulin. [B8] The method according to any one of [B1] to [B7], wherein the production culture medium is a culture medium containing no protein. [B9] The method according to any one of [B1] to [B8], wherein the production culture medium is a culture medium containing no serum. [B10] The method according to any one of [B1] to [B9], further comprising recovering the supernatant of the production culture medium after the culture 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 basal cell culture medium or a culture medium containing a basal cell culture medium supplemented with nutrients for cells. [B12] The method according to any one of [B1] to [B11], wherein the production culture medium is a basal cell culture medium or a basal cell culture medium supplemented with nutrients for cells. [B13] The method according to any one of [B1] to [B12], wherein the production culture medium is a basal cell culture medium supplemented with nutrients for cells, preferably amino acids. [B14] The method according to any one of [B1] to [B13], wherein the production culture medium does not contain a laminin fragment or a modified form thereof. [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 a growth culture medium for the cells. [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 growth 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 basal medium for cell culture supplemented with a growth factor. [B19] The method according to any one of [B2] to [B18], wherein the recovery culture medium is a basal medium for cell culture supplemented with a growth factor. [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 a laminin fragment or a variant thereof. [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 a cycle of culture in the production culture medium and culture in the recovery culture medium is repeated 2 to 10 times.

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

[0101] <4. Mesenchymal stem cell population and method for producing same> The present inventors have newly discovered that, according to the method described in the section <1. Method for producing expanded cells>, when mesenchymal stem cells are cultured in a proliferation medium in the presence of a laminin fragment that has integrin-binding activity, the expression of HLA-ABC and CD105, which are cell surface markers of mesenchymal stem cells, is reduced (see Example 4 below).

[0102] It is known in the art that mesenchymal stem cells are HLA-ABC positive and CD105 positive. Furthermore, in the art, cell surface marker positivity can be expressed by the value of the cell surface marker positivity rate. The cell surface marker positivity rate can be determined by a flow cytometer using a fluorescently labeled antibody of the cell surface marker, as described in the Examples below.

[0103] Therefore, according to one embodiment, there is provided a method for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a growth medium in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin-binding activity, to obtain expanded cells. The expanded cells obtained by this method have a reduced HLA-ABC positivity rate. The "HLA-ABC positivity rate" represents the proportion (%) 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] According to a preferred embodiment, there is provided 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 growth medium in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin-binding activity, to obtain expanded cells. The expanded cells obtained by this method have a reduced HLA-ABC positivity rate and a reduced CD105 positivity rate. The "HLA-ABC positivity rate" represents the proportion (%) of HLA-ABC-positive mesenchymal stem cells in the mesenchymal stem cell population, and the "CD105 positivity rate" represents the proportion (%) 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 that has 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 that has 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 "method for producing a mesenchymal stem cell population" can be carried out as explained in the section <1. Method for producing expanded cells>. In the section <1. Method for producing expanded cells>, the cell density at the start of culture (i.e., cell seeding density) is set to a seeding density lower than usual (0.002 to 2000 cells / cm). 2 However, in the above-mentioned "method for producing a mesenchymal stem cell population," it is not necessary to adopt a seeding density lower than usual, and a seeding density of 2000 cells / cm is used. 2That is, in the above-mentioned "method for producing a mesenchymal stem cell population," the cell density at the start of the expansion culture (i.e., cell seeding density) may be, for example, 2,001 to 1,000,000 cells / cm. 2 , preferably 5,000 to 20,000 cells / cm 2 Of course, in this method, a lower seeding density than usual (0.002 to 2000 cells / cm) can be used. 2 ) may also be adopted.

[0108] Mesenchymal stem cells with a low HLA-ABC positivity rate are less susceptible to the host's immune response (immune rejection) when administered as a therapeutic agent, and are therefore expected to have a high cell survival rate and a high therapeutic effect. It has been reported that cells with a low CD105 positivity rate have high levels of TGFb1 expression and suppress T cell proliferation, and are therefore expected to be effective in treating inflammatory diseases.

[0109] As used herein, the term "mesenchymal stem cells" encompasses mesenchymal stem cells before being cultured by the above-described method, as well as mesenchymal stem cells obtained by culturing by the above-described method. That is, as used herein, the term "mesenchymal stem cells" encompasses 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, as used herein, 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 same> The present inventors newly discovered that when mesenchymal stem cells were cultured in a proliferation culture medium in the presence of a laminin fragment with integrin-binding activity according to the method described in the section <2. Method for producing a cell product>, and then alternately cultured in a production culture medium and a recovery culture medium, and the supernatant of the production culture medium was collected, the amounts of cytokines, extracellular matrix, and exosomes in the culture supernatant increased with the number of production cultures (see Example 5 below).

[0111] Thus, one embodiment provides a method for producing a stem cell culture supernatant, comprising: culturing stem cells in a growth culture medium by adhesion culture in the presence of a culture substrate selected from a laminin fragment and its variants having integrin-binding activity to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product; and, after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells, and the method further comprises recovering 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] As used herein, 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 the culture, and can also be called a cell-derived culture supernatant. Cell culture supernatant also includes culture supernatant that has been subjected to sterilization or other treatments. Expressions such as "culture supernatant of stem cells" and "culture supernatant of mesenchymal stem cells" and "culture supernatant of specific cells" have the same meaning.

[0113] This method can be carried out as described in the section <2. Method for producing a cell product>. In the section <2. Method for producing a cell product>, the cell density at the start of the growth culture (i.e., cell seeding density) is set to a lower seeding density than usual (0.002 to 2000 cells / cm). 2 However, in the above-mentioned "method for producing stem cell culture supernatant," it is not necessary to adopt a seeding density lower than usual, and a seeding density of 2000 cells / cm is used. 2 That is, in this method, the cell density at the start of the expansion culture (i.e., the cell seeding density) may be, for example, 2,001 to 1,000,000 cells / cm. 2 , preferably 5,000 to 20,000 cells / cm 2Of course, in this method, a lower seeding density than usual (0.002 to 2000 cells / cm) can be used. 2 ) may also be adopted.

[0114] As described above, by repeating production culture and recovery culture alternately according to this method and recovering the supernatant of the production culture, the amounts of cytokines, extracellular matrix, and exosomes in the culture supernatant can be increased with each production culture. Therefore, in this method, the supernatant of the production culture is preferably recovered after the second or subsequent production culture in the production culture. More preferably, the supernatant of the production culture is recovered after the third or subsequent production culture in the production culture.

[0115] The supernatant of the production culture can be collected repeatedly as long as the amount of cell products, such as cytokines, secreted by the stem cells is maintained. That is, in this method, the number of times the production culture can be repeated is not particularly limited. For example, the supernatant of the production culture can be collected after the second to tenth production culture cycles in the production culture.

[0116] According to the above-mentioned "method for producing stem cell culture supernatant," it is possible to obtain stem cell culture supernatant containing large amounts of cell products such as cytokines, extracellular matrix, and exosomes.

[0117] Specifically, the above-described "method for producing stem cell culture supernatant" can produce a stem cell culture supernatant containing 5,000 pg / mL or more of hepatocyte growth factor (HGF) (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, 5,000 pg / mL or more, preferably 10,000 pg / mL or more, and more preferably 15,000 pg / mL or more of HGF. This culture supernatant contains, for example, 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. HGF is known to have angiogenic and wound healing effects.

[0118] Furthermore, the above-described "method for producing stem cell culture supernatant" can produce a stem cell culture supernatant containing 50 pg / mL or more of CD9 / CD63 EC domain fusion protein (see Figure 42). Hereinafter, this culture supernatant is also 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. The 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 a preferred embodiment, each of the culture supernatants according to the first embodiment and the second embodiment can 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 FIGS. 18 to 20 ). Each of the culture supernatants according to the first embodiment and the second embodiment can 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 embodiment and the second embodiment can further contain, for example, 3,000 to 1,000,000 pg / mL, preferably 4,000 to 1,000,000 pg / mL, and more preferably 6,000 to 1,000,000 pg / mL of MCP-1; for example, 1,000 to 1,000,000 pg / mL, preferably 2,000 to 1,000,000 pg / mL, and more preferably 4,000 to 1,000,000 pg / mL of GRO; and, for example, 5 to 1,000 μg / mL, preferably 6 to 1,000 μg / mL, and more preferably 8 to 1,000 μg / mL of fibronectin. MCP-1, GRO, and fibronectin are all known to have angiogenic and wound healing effects. Since GRO is also known as CXCL1, it is also referred to as GRO / CXCL1 in this specification.

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

[0121] Preferably, the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment do not contain at least one of insulin, transferrin, and albumin. It is more preferable that the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment do not contain any of insulin, transferrin, and albumin. In the production culture for obtaining a culture supernatant, a culture medium that does not contain the above-mentioned components is used as the production culture medium, thereby obtaining a culture supernatant that does not contain the above-mentioned components. Considering that the culture supernatant will be used as a therapeutic agent, it is desirable that it does not contain the above-mentioned components.

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

[0123] Furthermore, it is preferable that the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment each contain no xenogeneic components. Here, "xenogeneic" refers to a species different from the biological species of the 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 xenogeneic components.

[0124] Each of the culture supernatants according to the first embodiment and the second embodiment 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). Each of the culture supernatants according to the first embodiment and the second embodiment more preferably contains IL-1α, IL-1β, and TNF-α in an amount of 0 to 15 pg / mL. Because these cytokines are known to be factors that cause inflammatory symptoms, it is desirable that they are not contained in large amounts in the culture supernatants.

[0125] Each of the culture supernatants according to the first embodiment and the second embodiment preferably contains 20 or more types of cytokines, each of which preferably has a concentration of 10 pg / mL or more. In the Examples described below, the culture supernatant collected after the third production culture contained cytokines such as 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 (40 kDa subunit of interleukin-12), IP-10 (interferon gamma-induced protein-10), and MIP-1α (macrophage inflammatory cytokine). It has been demonstrated that the 20 or more cytokines contained in the serum contain cytokines such as erythrocyte protease inhibitor (erythrocyte protease inhibitor) and erythrocyte protease inhibitor (erythrocyte protease inhibitor) and that each of these cytokines has a concentration of 10 pg / mL or more (see Figures 16-19, 21-29, 33-41). The concentration of each cytokine among the 20 or more cytokines varies depending on the type of cytokine, but is, for example, 1,000,000 pg / mL or less.

[0126] Each of the culture supernatants according to the first embodiment and the second embodiment is preferably a culture supernatant of mesenchymal stem cells. Each of the culture supernatants according to the first embodiment and the second embodiment is more preferably a culture supernatant of umbilical cord-derived mesenchymal stem cells. The culture supernatant of mesenchymal stem cells can contain larger amounts of cytokines, extracellular matrix, and exosomes. The culture supernatant of umbilical cord-derived mesenchymal stem cells can contain particularly large amounts of cytokines, extracellular matrix, and exosomes.

[0127] Although the examples described below describe the culture supernatant of mesenchymal stem cells, it is known that stem cells other than mesenchymal stem cells, such as pluripotent stem cells such as ES cells and iPS cells, and somatic stem cells such as neural stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells, also secrete cytokines, extracellular matrix, and exosomes into the culture medium. Therefore, even when the above-mentioned "method for producing stem cell culture supernatant" is performed using stem cells other than mesenchymal stem cells, such as pluripotent stem cells such as ES cells and iPS cells, or somatic stem cells such as neural stem cells, skin stem cells, hepatic 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-mentioned "method for producing stem cell culture supernatant" is performed using cells other than stem cells that are known to secrete cytokines, extracellular matrix, and exosomes into the culture medium, it is possible to obtain a culture supernatant containing large amounts of cytokines, extracellular matrix, and exosomes, just like the "stem cell culture supernatant." Therefore, the above-mentioned "method for producing stem cell culture supernatant" can be expanded to include a "method for producing cell culture supernatant," and the "stem cell culture supernatant" can be expanded to include a "cell culture supernatant."

[0129] <6. Therapeutic Agent> The present inventors have newly discovered that when mesenchymal stem cells are cultured in a proliferation medium in the presence of laminin fragments having integrin binding activity according to the method described in the section <1. Method for Producing Proliferated Cells>, the obtained mesenchymal stem cells can significantly increase blood flow in the ischemic hind limbs of a rat hind limb ischemia model (see Example 6 below).

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

[0131] Another aspect provides a therapeutic agent containing the culture supernatant of the present invention. 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, followed by culturing them in a production culture medium, and recovering the supernatant of the production culture medium, according to the method described in Section <2. Method for Producing a Cell Product>. The culture supernatant of the present invention can also be obtained by culturing mesenchymal stem cells in a proliferation culture medium in the presence of a laminin fragment having integrin-binding activity, followed by repeatedly culturing them in a production culture medium and a recovery culture medium, and recovering the supernatant of the production culture medium, according to the method described in Section <2. Method for Producing a Cell Product>. As described above, the culture supernatant of the present invention is characterized by containing larger amounts of cytokines, extracellular matrix, and exosomes than 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, a therapeutic agent comprising the mesenchymal stem cell population of the present invention or the culture supernatant of the present invention can be used to treat diseases for which mesenchymal stem cells are known to have a therapeutic effect, diseases for which mesenchymal stem cell culture supernatant is known to have a therapeutic effect, as well as diseases for which cytokines are known to have a therapeutic effect, diseases for which extracellular matrices are known to have a therapeutic effect, and diseases for which exosomes are known to have a therapeutic effect.

[0133] For example, therapeutic agents comprising the mesenchymal stem cell population of the present invention or the culture supernatant of the present invention can be 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; and sarcopenia associated with aging, because 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, since TGF-1b, IL-4, IL-10, and IL-13 are known to be cytokines with anti-inflammatory effects, therapeutic agents containing the mesenchymal stem cell population of the present invention or the culture supernatant of the present invention can be used to treat arthritis such as rheumatism, herniated disc, and osteoarthritis; inflammatory diseases such as nephritis, keratitis, and cytokine storm; and psychiatric disorders such as autism and insomnia, which are thought to be caused in part by neuroinflammation.

[0135] Furthermore, since IL-7, IL-15, GM-CSF, and G-CSF are thought to be involved in immune regulation, therapeutic agents containing the mesenchymal stem cell population of the present invention or the 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; and cancer diseases.

[0136] When a mesenchymal stem cell population is used as an active ingredient in a therapeutic agent, the mesenchymal stem cells of the present invention can continuously secrete cell products such as cytokines, and therefore a long-term therapeutic effect can be expected. On the other hand, when a mesenchymal stem cell culture supernatant is used as an active ingredient in a therapeutic agent, the culture supernatant of the present invention contains a large amount of cell products such as cytokines, and therefore a sufficient therapeutic effect can be expected.

[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 preparations, preferably injectable or topical liquid preparations. The therapeutic agent may be diluted with a pharmaceutically acceptable medium. The pharmaceutically acceptable medium is, for example, a mesenchymal stem cell culture medium or an infusion preparation. The therapeutic agent may contain additives to increase storage stability, isotonicity, absorbability, and / or viscosity. Alternatively, when the therapeutic agent contains a mesenchymal stem cell population, it may be in the form of a cell sheet.

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

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

[0140] 7. Preferred Embodiments Preferred embodiments of the present invention are summarized below. 7-1. Mesenchymal stem cell population and method for producing same [C1] A method for producing a mesenchymal stem cell population with a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a growth medium in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin-binding activity to obtain proliferated cells. [C2] The method described in [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 described in [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 described in [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 proliferation culture medium is a proliferation 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 proliferation culture medium is a culture medium containing a basal medium for cell culture supplemented with a growth factor. [C9] The method according to any one of [C1] to [C8], wherein the proliferation culture medium is a basal medium for cell culture supplemented with a growth factor. [C10] The method according to any one of [C1] to [C9], wherein the culturing is carried out until the cells reach a confluent state.

[0142] [C11] The culture is 500 cm 2 [C12] The method according to any one of [C1] to [C10], wherein the culture is carried out in a culture vessel having a bottom area of ​​500 to 10,000 cm or more. 2 [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 a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a 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 that has 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 a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a 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 concentration of the laminin fragment or its modified product in the growth culture medium is 0.1% or more per 1 cm of culture area of ​​the culture vessel. 2 [C29] 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 cm of culture area of ​​the culture vessel. 2 [C30] 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 cm of culture area of ​​the culture vessel. 2[C31] The method according to any one of [C1] to [C30], wherein the decreased 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 having a proportion of HLA-ABC-positive mesenchymal stem cells of 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 according to [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. [D5] A mesenchymal stem cell population obtainable by the method according to any one of [C1] to [C31]. [D6] The mesenchymal stem cell population according to any one of [D1] to [D4], wherein the mesenchymal stem cell population is obtained by the method according to 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 stem cell culture supernatant, comprising: culturing stem cells in a growth culture medium by adhesion culture in the presence of a culture substrate selected from a laminin fragment and its variant having integrin binding activity to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product; and, after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells, and the method further comprises recovering the supernatant of the production culture medium after the culture in the production culture medium. [E2] The method described in [E1], wherein the supernatant of the production culture medium is recovered after the second or subsequent culture in the production culture medium, preferably after the third or subsequent culture 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, or adipose stem cells; or pluripotent stem cells such as induced pluripotent stem cells (iPS cells) or 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 hepatic stem cells, human muscle stem cells, or human adipose stem cells; or human pluripotent stem cells such as human induced pluripotent stem cells (human iPS cells) or 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 proliferation culture medium is a proliferation culture medium containing a protein that promotes proliferation of the stem cells. [E13] The method according to any one of [E1] to [E12], wherein the proliferation culture medium is a culture medium containing a basal cell culture medium supplemented with a growth factor. [E14] The method according to any one of [E1] to [E13], wherein the proliferation culture medium is a basal cell culture medium supplemented with a growth factor. [E15] The method according to any one of [E1] to [E14], wherein the culturing in the proliferation culture medium is carried out until the cells reach a confluent state.

[0150] [E16] The culture in the growth medium is 500 cm 2 [E17] The method according to any one of [E1] to [E15], wherein the culture is carried out in a culture vessel having a bottom area of ​​500 to 10,000 cm or more. 2[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 a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a 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 a laminin 511 E8 fragment, a laminin 521 E8 fragment, a laminin 411 E8 fragment, a laminin 421 E8 fragment, a laminin 332 E8 fragment, a laminin 311 E8 fragment, a laminin 321 E8 fragment, a laminin 211 E8 fragment, a laminin 221 E8 fragment, a laminin 213 E8 fragment, a laminin 111 E8 fragment, or a 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 [E23] to [E30], wherein the concentration of the laminin fragment or its modified product in the growth culture medium is 1 / 2 times the concentration of the laminin fragment or its modified product per cm of culture area of ​​the culture vessel. 2 [E34] 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 cm of the culture area of ​​the culture vessel. 2 [E35] 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 cm of culture area of ​​the culture vessel. 2 The method according to any one of [E1] to [E34], wherein the amount is 0.05 μg to 0.25 μg per unit area.

[0154] [E36] The method according to any one of [E1] to [E35], wherein the production culture medium is a culture medium containing no xenogeneic components. [E37] The method according to any one of [E1] to [E36], wherein the production culture medium is a culture medium containing no cytokines or insulin. [E38] The method according to any one of [E1] to [E37], wherein the production culture medium is a culture medium containing no protein. [E39] The method according to any one of [E1] to [E38], wherein the production culture medium is a culture medium containing no serum. [E40] The method according to any one of [E1] to [E39], wherein the production culture medium is a culture medium containing a basal cell culture medium supplemented with nutrients for the cells.

[0155] [E41] The method according to any one of [E1] to [E40], wherein the production culture medium is a basal cell culture medium or a basal cell culture medium supplemented with nutrient components for cells. [E42] The method according to any one of [E1] to [E41], wherein the production culture medium is a basal cell culture medium supplemented with nutrient components for cells, preferably amino acids. [E43] The method according to any one of [E1] to [E42], wherein the production culture medium does not contain a laminin fragment or a modified form thereof. [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 a proliferation culture medium for 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 basal cell culture medium supplemented with a growth factor. [E48] The method according to any one of [E1] to [E47], wherein the recovery culture medium is a basal cell culture medium supplemented with a growth factor. [E49] The method according to any one of [E1] to [E48], wherein the recovery culture medium has the same composition as the proliferation culture medium. [E50] The method according to any one of [E1] to [E49], wherein the recovery culture medium does not contain a laminin fragment or a modified form thereof. [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] A stem cell culture supernatant containing 5,000 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] A stem cell culture supernatant containing 50 pg / mL or more of CD9 / CD63 EC domain fusion protein. [F5] The culture supernatant according to [F4], wherein the culture supernatant contains 100 pg / mL or more, preferably 200 pg / mL or more of CD9 / CD63 EC domain fusion protein.

[0158] [F6] The culture supernatant according to [F4] or [F5], wherein the culture supernatant contains 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, and more preferably 200 to 100,000 pg / mL, of the CD9 / CD63 EC domain fusion protein. [F7] The culture supernatant according to any one of [F1] to [F3], wherein the culture supernatant further contains 50 pg / mL or more of the 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 the CD9 / CD63 EC domain fusion protein. [F9] The culture supernatant according to any one of [F1] to [F3], further containing 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, more preferably 200 to 100,000 pg / mL of CD9 / CD63 EC domain fusion protein. [F10] The culture supernatant according to any one of [F1] to [F9], further containing 3,000 pg / mL or more of MCP-1, 1,000 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], further containing MCP-1 at 4000 pg / mL or more, preferably 6000 pg / mL or more; GRO at 2000 pg / mL or more, preferably 4000 pg / mL or more; and fibronectin at 6 μg / mL or more, preferably 8 μg / mL or more. [F12] The culture supernatant according to any one of [F1] to [F9], further containing 3,000 to 1,000,000 pg / mL, preferably 4,000 to 1,000,000 pg / mL, and more preferably 6,000 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, and more preferably 4,000 to 1,000,000 pg / mL of GRO; and 5 to 1,000 μg / mL, preferably 6 to 1,000 μg / mL, and more preferably 8 to 1,000 μg / mL of fibronectin. [F13] The culture supernatant according to any one of [F1] to [F12], further containing 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], further containing 300 pg / mL or more, preferably 500 pg / mL or more of TGF-1b, 10 pg / mL or more, preferably 20 pg / mL or more of IL-4, and 10 pg / mL or more, preferably 12 pg / mL or more of IL-10. [F15] The culture supernatant according to any one of [F1] to [F12], further containing 200 to 100,000 pg / mL, preferably 300 to 100,000 pg / mL, and more preferably 500 to 100,000 pg / mL of TGF-1b; 5 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, and 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, and 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 any of insulin, transferrin, and albumin. [F18] The culture supernatant according to any one of [F1] to [F17], wherein the culture supernatant does not contain a 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.

[0161] [F21] The culture supernatant according to any one of [F1] to [F20], wherein the culture supernatant contains 20 or more cytokines, each of which 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 a culture supernatant of somatic stem cells such as mesenchymal stem cells, neural stem cells, skin stem cells, hepatic stem cells, muscle stem cells, or adipose stem cells; or a culture supernatant of pluripotent stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). [F23] The culture supernatant according to any one of [F1] to [F22], wherein the culture supernatant is a culture supernatant of mesenchymal stem cells. [F24] The culture supernatant according to any one of [F1] to [F23], wherein the culture supernatant is a culture supernatant of umbilical cord-derived mesenchymal stem cells, a culture supernatant of bone marrow-derived mesenchymal stem cells, a culture supernatant of adipose-derived mesenchymal stem cells, a culture supernatant of placenta-derived mesenchymal stem cells, or a 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 a culture supernatant of umbilical cord-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells, preferably a 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 a culture supernatant of human mesenchymal stem cells. [F27] The culture supernatant according to any one of [F1] to [F24], wherein the culture supernatant is a culture supernatant of 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. [F28] The culture supernatant according to any one of [F1] to [F25], wherein the culture supernatant is a culture supernatant of human umbilical cord-derived mesenchymal stem cells or human adipose-derived mesenchymal stem cells, preferably human umbilical cord-derived mesenchymal stem cells. [F29] A culture supernatant obtainable by the method according to any one of [E1] to [E52]. [F30] The culture supernatant according to any one of [F1] to [F28], wherein the culture supernatant is obtained by the method according to any one of [E1] to [E52].

[0163] <7-3. Therapeutic Agents> [G1] A therapeutic agent comprising the mesenchymal stem cell population described in any one of [D1] to [D6] or the culture supernatant described in any one of [F1] to [F30]. [G2] A therapeutic agent comprising the mesenchymal stem cell population described in any one of [D1] to [D6]. [G3] A therapeutic agent comprising the culture supernatant described in any one of [F1] to [F30]. [G4] The therapeutic agent according to any one of [G1] to [G3], wherein the therapeutic agent is for treating an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; wounds such as epithelial wounds or burns; sarcopenia associated with aging; arthritis such as rheumatism, herniated disc, or osteoarthritis; inflammatory diseases such as nephritis, keratitis, or cytokine storm; psychiatric disorders such as autism or insomnia, which are thought to be caused in part by neuroinflammation; immune diseases such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, or autoimmune disease; 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] [G6] A method for treating an injury or disease, comprising administering to a subject the mesenchymal stem cell population described in any one of [D1] to [D6] or the culture supernatant described in any one of [F1] to [F30]. [G7] A method for treating an injury or disease, comprising administering to a subject the mesenchymal stem cell population described in any one of [D1] to [D6]. [G8] A method for treating an injury or disease, comprising administering to a subject the culture supernatant described in any one of [F1] to [F30]. [G9] The method according to any one of [G6] to [G8], wherein the injury or disease is an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; a wound such as epithelial wound or burn; sarcopenia associated with aging; arthritis such as rheumatoid arthritis, herniated disc, or osteoarthritis; an inflammatory disease such as cytokine storm, nephritis, or keratitis; a psychiatric disorder such as autism or insomnia, which is thought to be caused in part by neuroinflammation; an immune disease such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, or autoimmune disease; or a cancer. [G10] The therapeutic agent according to any one of [G6] to [G9], wherein the injury or disease 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 the mesenchymal stem cell population described in any one of [D1] to [D6] or the culture supernatant described in any one of [F1] to [F30] for the manufacture of a therapeutic agent. [G13] Use of the mesenchymal stem cell population described in any one of [D1] to [D6] for the manufacture of a therapeutic agent. [G14] Use of the culture supernatant described in any one of [F1] to [F30] for the manufacture of a therapeutic agent. [G15] The use according to any one of [G12] to [G14], wherein the therapeutic agent is for treating an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; wounds such as epithelial wounds or burns; sarcopenia associated with aging; arthritis such as rheumatoid arthritis, herniated disc, or osteoarthritis; inflammatory diseases such as cytokine storm, nephritis, or keratitis; psychiatric disorders such as autism or insomnia, which are thought to be caused in part by neuroinflammation; immune diseases such as GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, or autoimmune disease; or cancer. [G16] The therapeutic agent according to any one of [G12] to [G15], 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.

[0166] Example 1: Expansion culture (1) Method Stem cell expansion culture was performed in Example 1. Umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in a cryopreservation solution from Stem Cell Banker (Zenoac) were used as stem cells.

[0167] Experiment 1-1 (Control) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were seeded at a cell density of 5x10e3 (seeding density 10 cells / cm 2 ), 5x10e4 cells (seeding density 100 cells / cm 2 ), 5x10e5 cells (seeding density 1000 cells / cm 2 ), and 5x10e6 cells (seeding density 10000 cells / cm 2The cells were then suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.), and the resulting cell suspension was seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). Four flasks were seeded for each condition.

[0168] During the expansion culture, the medium was changed every 5 days. Five days (day 5), 10 days (day 10), 15 days (day 15), and 20 days (day 20) after seeding, each flask was irradiated with TrypLE TM The cells were treated with Select (Thermo Fisher Scientific) for 10 to 20 minutes to disperse them into single cells, and then the number of cells was counted using a cell counter.

[0169] The number of proliferated cells is shown in Figure 1. In Figure 1, the vertical axis of the graph shows the counted number of cells divided by the flask area. 2 FIG. 2 shows a micrograph of the cells after 20 days of culture.

[0170] Experiment 1-2 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were cultured at a cell density of 5x10e3 (seeding density 10 cells / cm 2 ), 5x10e4 cells (seeding density 100 cells / cm 2 ), 5x10e5 cells (seeding density 1000 cells / cm 2 ), and 5x10e6 cells (seeding density 10000 cells / cm 2 The cells were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.), iMatrix-511 laminin fragments (Nippi Corporation) were added at 50 μl / 100 ml, and the resulting cell suspension was seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.). Four flasks were seeded for each condition.

[0171] During the expansion culture, the medium was changed every 5 days. Five days after seeding, iMatrix-511 laminin fragments were added again at 50 μl / 100 ml. However, laminin fragments were not added on days 10, 15, and 20 after seeding.

[0172] Five days (day 5), 10 days (day 10), 15 days (day 15), and 20 days (day 20) after seeding, each flask was treated with TrypLE. TM The cells were treated with Select (Thermo Fisher Scientific) for 10 to 20 minutes to disperse them into single cells, and then the number of cells was counted using a cell counter.

[0173] The number of proliferated cells is shown in Figure 3. In Figure 3, the vertical axis of the graph shows the counted number of cells divided by the flask area. 2 FIG. 4 shows a micrograph of the cells after culturing for 20 days.

[0174] Experiment 1-3 (Reference Example) Umbilical cord-derived mesenchymal stem cells (UCMSC) were cultured at a cell density of 5x10e3 (seeding density 10 cells / cm 2 ), 5x10e4 cells (seeding density 100 cells / cm 2 ), 5x10e5 cells (seeding density 1000 cells / cm 2 ), and 5x10e6 cells (seeding density 10000 cells / cm 2 The cells were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Corporation) and seeded onto a gelatin-coated peel-off T512 flask (Sumitomo Bakelite Co., Ltd.). The gelatin coating was performed by treating with gelatin solution (StemSure 0.1 w / v% Gelatin Solution, Fujifilm Wako Pure Chemical Corporation) for 2 hours.

[0175] During the expansion culture, the medium was changed every 5 days. Five days (day 5), 10 days (day 10), 15 days (day 15), and 20 days (day 20) after seeding, each flask was irradiated with TrypLE TM The cells were treated with Select (Thermo Fisher Scientific) for 10 to 20 minutes to disperse them into single cells, and then the number of cells was counted using a cell counter.

[0176] The number of proliferated cells is shown in Figure 5. In Figure 5, the vertical axis of the graph shows the value obtained by dividing the counted number of cells by the flask area. 2 FIG. 6 shows a micrograph of the cells after culturing for 20 days.

[0177] (2) Results When umbilical cord-derived mesenchymal stem cells (UCMSCs) proliferated and reached confluence in a T512 flask, the number of cells reached approximately 3 x 10e7 (i.e., approximately 60,000 cells / cm). 2 ) was recovered. Therefore, in Figures 1, 3 and 5, approximately 60,000 cells / cm 2 indicates a confluent state.

[0178] As can be seen from the results in Figure 1, in the absence of laminin fragments, stem cells were cultured at a density of 10,000 cells / cm. 2 When stem cells are seeded and cultured at a seeding density of 1000 cells / cm, they can be grown to a confluent state. 2 It was shown that when the cells were seeded and cultured at a cell density of less than 10 cells / cm, cell proliferation stopped and they could not be grown to a confluent state. Furthermore, the micrographs in Figure 2 show that in the absence of laminin fragments, stem cells were cultured at a density of 10 cells / cm. 2 When cells were seeded and cultured at a low cell density, the cells did not grow until they reached confluence, and growth stopped at the colony stage.

[0179] From the results in Figure 3, in the presence of laminin fragments, stem cells were cultured at 1000 cells / cm 2 It was shown that stem cells could be grown to a confluent state even when seeded and cultured at a low cell density of 10 cells / cm or less. Furthermore, the micrograph in Figure 4 shows that in the presence of laminin fragments, stem cells could be grown at a density of 10 cells / cm. 2 It was shown that cells can grow to a confluent state even when seeded and cultured at a low cell density.

[0180] Furthermore, the present inventors have used the same method as in Experiment 1-2 to culture umbilical cord-derived mesenchymal stem cells (UCMSCs) at a density of 2 cells / cm. 2This demonstrates that cells can be grown to confluence even when seeded at a cell density of 1000 kJ / well and cultured in growth medium containing iMatrix-511 laminin fragments.

[0181] From the results in Figure 5, in the presence of gelatin, stem cells were cultured at a density of 10,000 cells / cm 2 When stem cells are seeded and cultured at a seeding density of 1000 cells / cm, they can be grown to a confluent state. 2 It was shown that when the cells were seeded and cultured at a cell density below 10 cells / cm, cell proliferation stopped and they could not be grown to a confluent state. Furthermore, the micrographs in Figure 6 show that in the presence of gelatin, stem cells were cultured at a density of 10 cells / cm. 2 When cells were seeded and cultured at a low cell density, the cells did not grow until they reached confluence, and growth stopped at the colony stage.

[0182] These results demonstrate that when stem cells are cultured in the presence of laminin fragments, they can grow to confluence even when seeded at a low cell density.

[0183] Example 2 Production Culture (1) Method In Example 2, stem cells were cultured for proliferation and then cultured for production. Umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in a cryopreservation solution from Stem Cell Banker (Zenoac) were used as stem cells.

[0184] Experiment 2-1 (Comparative Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were seeded at a density of 5x10e5 (1000 cells / cm 2 ) were suspended 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 a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).

[0185] During the expansion culture, the medium was changed every 5 days. On day 15 after seeding, the medium was replaced twice with PBS to wash out any remaining medium. Then, 120 ml of protein-free medium (hereinafter referred to as MSC medium A) based on DMEM / F12 medium supplemented with 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). MSC medium A was added and the culture (production culture) was continued for 3 days. After 3 days of culture, two images of the cells were taken with an Olympus microscope. The two images were taken at different positions within 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 seeded at a density of 5x10e5 (1000 cells / cm 2 ) were suspended in 100 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B), iMatrix-511 laminin fragment (Nippi Corporation) was added at 50 μl / 100 ml, and the resulting cell suspension was seeded into a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).

[0187] During the expansion culture, the medium was changed every 5 days. Five days after seeding, iMatrix-511 laminin fragments were added again at 50 μl / 100 ml. However, no laminin fragments were added 10 days after seeding.

[0188] On day 15 after seeding, the medium was replaced twice with PBS to wash away any remaining medium. Then, 120 ml of protein-free medium (hereinafter referred to as MSC medium A) based on DMEM / F12 medium supplemented with 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). MSC medium A was added and the cells were cultured for three days (production culture). After three days of culture, two images of the cells were taken with 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 arrows indicate the locations of cell clumps in Figure 7A. In Figure 7B, most of the cells had detached and no longer remained. In both images of Figures 8A and 8B, however, the cells remained attached and intact.

[0190] These results indicate that if stem cells are cultured and expanded in the presence of laminin fragments, and then the resulting expanded cells are cultured in a protein-free culture medium that does not contain any exogenous components, the cells will not detach from the culture vessel during the culture process and will remain adherent.

[0191] Example 3 Long-term Production Culture (1) Method In Example 3, stem cells were cultured for proliferation, followed by long-term production culture. The culture process performed in Example 3 is shown in Figure 9. Umbilical cord-derived mesenchymal stem cells (UCMSCs) cryopreserved in a cryopreservation solution from Stem Cell Banker (Zenoac) were used as stem cells.

[0192] Experiment 3-1 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were suspended in 100 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B) at a cell count of 5 x 10e5 (seeding density 1000 cells / cm2), and iMatrix-511 laminin fragments (Nippi Corporation) were added at 50 μl / 100 ml. The resulting cell suspension was seeded into a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).

[0193] During the expansion culture, the medium was changed every 5 days. Five days after seeding, iMatrix-511 laminin fragments were added again at 50 μl / 100 ml. However, no laminin fragments were added 10 days after seeding.

[0194] Fifteen days after seeding (day 15), the cells were confirmed to have reached confluence, and the medium was replaced twice with PBS to wash away any remaining medium. Then, 120 ml of protein-free medium (hereinafter referred to as MSC medium A) based on DMEM / F12 medium supplemented with 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). After adding MSC medium A, the cells were cultured for four days. This culture is shown as the "first production culture" in Figure 9.

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

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

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

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

[0199] After the third production culture, the culture supernatant of MSC medium A was collected. The 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, production culture was performed three times in total, and culture supernatant was collected and cytokine analysis was performed three times in total.

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

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

[0203] In this experiment, since laminin fragments were not added, the cells began to detach from the culture vessel three days after replacement with MSC medium A. Therefore, the culture supernatant was collected two days after replacement with MSC medium A.

[0204] (2) Results The quantification results of cytokines are shown in Figures 10 to 12. The graphs in Figures 10 to 12 show, from left to right, 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 an example of the present invention, the amount of cytokines in the culture supernatant recovered after the third production culture in an example of the present invention, and the amount of cytokines in the culture supernatant recovered in the comparative example.

[0205] The results of Figures 10 to 12 demonstrate that long-term production culture according to the method of the present invention can produce cytokines in amounts comparable to or greater than those in the comparative example, and that the production amount does not decrease or even increases with repeated production culture. VEGF and HGF are cytokines with angiogenic activity. While the production amount of VEGF was slightly lower than that of the comparative example in some cases, the absolute amount of VEGF produced was large, so it can be said that sufficient amounts were produced. HGF production tended to increase with repeated production culture. IL-7 is a cytokine involved in immune cell activation and inflammation. IL-7 production tended to increase with repeated production culture.

[0206] In Example 3, production culture was performed three times in total, and culture supernatant was collected and cytokine analysis was performed three times in total. However, the present inventors have performed production culture six times in total to demonstrate that cytokines can be produced.

[0207] These results reveal the following: After culturing and expanding stem cells in the presence of laminin fragments, the resulting expanded cells can be cultured in a protein-free medium that does not contain any exogenous components, allowing production culture to be performed while maintaining the adherent state of the expanded cells. Furthermore, after this production culture, recovery culture can be performed while maintaining the adherent state of the cells, thereby restoring the cells' ability to produce cell products. This allows production culture to be performed multiple times while maintaining the adherent state of the cells, resulting in the long-term production of large amounts of cell products using a simple method.

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

[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 cultured at a cell density of 5x10e5 (seeding density 1000 cells / cm 2 ) and suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.), iMatrix-511 laminin fragment (Nippi Corporation) was added at 50 μl / 100 ml, 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, or 3 weeks, and adipose-derived mesenchymal stem cells were cultured for 1 or 2 weeks.

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

[0211] Umbilical cord-derived mesenchymal stem cells were seeded onto 24-well plates (Corning) in the presence of laminin fragments and cultured for one week. Afterwards, the mesenchymal stem cells that adhered to the 24-well plates were immunostained with PE-conjugated antibodies against the cell surface markers CD73, CD90, CD105, and HLA-ABC, and bright-field and fluorescent images were taken using a fluorescence microscope (Keyence).

[0212] Experiment 4-2 (Comparative Example) Umbilical cord-derived mesenchymal stem cells (UCMSCs) were cultured at a cell density of 5x10e5 (seeding density 1000 cells / cm 2 The cells were suspended in 100 ml of MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) and seeded into peel-off T512 flasks (Sumitomo Bakelite Co., Ltd.) without adding iMatrix-511 laminin fragments. Culture was carried out in the same manner as in Experiment 4-1, except that laminin fragments were not added.

[0213] After culturing, the positivity rates of cell surface markers CD44, CD73, CD90, CD105, and HLA-ABC were analyzed using the same procedures as in Experiment 4-1. The expression of cell surface markers CD73, CD90, CD105, and HLA-ABC was also confirmed using immunostaining images using the same procedures as in Experiment 4-1.

[0214] (2) Results Figure 13 shows the positive rate of cell surface markers for umbilical cord-derived mesenchymal stem cells when cultured in the presence of laminin fragments, Figure 14 shows the positive rate of cell surface markers for adipose-derived mesenchymal stem cells when cultured in the presence of laminin fragments, and Figure 15 shows the positive rate of cell surface markers for umbilical cord-derived mesenchymal stem cells when 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 positivity rates of CD105 and HLA-ABC were significantly reduced, but the positivity rates of markers other than CD105 and HLA-ABC were not reduced.On the other hand, when umbilical cord-derived mesenchymal stem cells were cultured in the absence of laminin fragments, the positivity rates of CD105 and HLA-ABC were not significantly reduced.

[0216] When umbilical cord-derived mesenchymal stem cells were cultured in the presence of laminin fragments, the immunostaining images showed that almost all cells were positive for CD73 and CD90, as was the case with flow cytometry. However, CD105- and HLA-ABC-positive cells were rarely observed. Because the immunostaining images were taken without treating the mesenchymal stem cells with protease, these results demonstrate that the protease treatment performed for flow cytometry analysis did not reduce the expression of CD105 or HLA-ABC.

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

[0218] (1) Methods The amounts of cytokines and extracellular matrix contained in the culture supernatant were analyzed using an ELISA analysis 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-1b, 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 also analyzed as an extracellular matrix. Furthermore, the amount of exosomes contained in the culture supernatant was analyzed using a CD9 / CD63 ELISA kit (Cosmo Bio) using the exosome marker protein (CD9 / CD63 fusion protein) as an indicator. As used herein, the amounts of cytokines, extracellular matrix, and exosome marker proteins refer to values ​​measured by ELISA using specific antibodies.

[0219] (2) Results In this example, as in Example 3, the example of the present invention was subjected to growth culture in the presence of laminin fragments, whereas the comparative example was subjected to growth culture in the absence of laminin fragments. Therefore, in the example of the present invention, multiple cycles of production culture were possible, whereas in the comparative example, the cells began to detach from the culture vessel during the first cycle of production culture, and therefore only one cycle of production culture was possible.

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

[0221] 16 to 42, "1st" indicates the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered after the first production culture (4 days) in the examples of the present invention, "2nd" indicates the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered after the second production culture (4 days) in the examples of the present invention, and "3rd," "4th," and "5th" have similar meanings. Also, in Figures 16 to 42, "Comparative Example" indicates the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered 2 days after the start of the first production culture in the comparative example.

[0222] 16 to 42, when the production culture was repeatedly performed according to the method of the present invention and the culture supernatant was repeatedly collected, the amount 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) tended to increase. Therefore, it was shown that when the production culture was repeatedly performed according to the method of the present invention and the culture supernatant was repeatedly collected, a culture supernatant containing a large amount of cytokines, a large amount of extracellular matrix, and a large amount of exosomes can be obtained.

[0223] As demonstrated in this example, when mesenchymal stem cell conditioned media is obtained by culturing in a protein-free medium without any exogenous components, all of the proteins contained in the conditioned media are derived from mesenchymal stem cells, making it advantageous for use as a therapeutic agent. Furthermore, repeated collection of mesenchymal stem cell conditioned media can increase the content of cytokines such as HGF, fibronectin, and exosomes, making it advantageous in that these proteins and exosomes are expected to have therapeutic effects. Furthermore, mesenchymal stem cell conditioned media is advantageous in that the content of IL-1α, IL-1β, and TNF-α, which are known to cause inflammation, does not increase with repeated collection of the conditioned media, resulting in a relatively low content of inflammatory cytokines.

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

[0225] (1) Methods <Preparation of rats for the hindlimb ischemia model> Twelve-week-old male Sprague Dawley (SD) rats (purchased from CLEA Japan, Osaka, Japan) were used. 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. Hindlimb ischemia was induced by exposing and ligating the external iliac artery and internal iliac vein, the saphenous artery and vein just below the ankle, and all of their branches, followed by excision of all of the ligated vessels.

[0226] Three days after induction of hindlimb ischemia, blood flow was measured using laser Doppler perfusion imaging (LDPI). To ensure successful induction of hindlimb ischemia and to exclude rats with significant self-regenerative capacity, allowing evaluation of the actual effects of cell therapy, only rats with a relative blood flow rate (%) of 60% or less in the ischemic hindlimb relative to the non-ischemic hindlimb were enrolled for treatment. Enrolled rats were randomly assigned to one of three groups: umbilical cord-derived mesenchymal stem cells (UCMSCs), adipose-derived mesenchymal stem cells (ADMSCs), or DMEM / F12.

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

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

[0229] <Blood flow measurement> Blood flow in the lower limbs was assessed using a Moor LDI 2.0 system (Moor instruments, Devon, UK). Blood flow was measured 14 days after the induction of lower limb ischemia. When measuring blood flow, rats were anesthetized with isoflurane inhalation. 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 of the lower limb blood flow ratio are shown in Figure 43. In Figure 43, "Control" represents the group administered with DMEM / F12 medium, "UCMSC laminin-" represents the group administered with umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragments, "UCMSC laminin+" represents the group administered with umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragments, "ADMSC laminin-" represents the group administered with adipose-derived mesenchymal stem cells cultured in the absence of laminin fragments, and "ADMSC laminin+" represents the group administered with adipose-derived mesenchymal stem cells cultured in the presence of laminin fragments. In Figure 43, error bars represent standard error.

[0231] The blood flow ratio of "UCMSC laminin+" and "ADMSC laminin+" was significantly increased compared to the control, and a t-test confirmed a significant difference from the control. On the other hand, the blood flow ratio of "UCMSC laminin-" and "ADMSC laminin-" tended to be slightly increased compared to the control, but the t-test did not confirm a significant difference from the control.

[0232] These results demonstrate that mesenchymal stem cells obtained according to the method of the present invention are effective as a therapeutic agent for lower limb ischemia. This therapeutic effect is believed to be brought about by the cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells, and therefore the mesenchymal stem cell culture supernatant obtained according to the method of the present invention is also believed to have a similar therapeutic effect. Furthermore, because this therapeutic effect is believed to be brought about by the cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells, the mesenchymal stem cells and mesenchymal stem cell culture supernatant obtained according to the method of the present invention are also believed to have a therapeutic effect against diseases for which cytokines are known to have a therapeutic effect, diseases for which extracellular matrix is ​​known to have a therapeutic effect, and diseases for which exosomes are known to have a therapeutic effect.

Claims

1. 0.002-2000 cells / cm 2 and culturing the cells in a growth medium by adhesion culture in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity, thereby growing the cells.

2. The method of claim 1, wherein the culturing is carried out in a growth medium containing the culture substrate.

3. The method of claim 1 or 2, wherein the cells are stem cells.

4. The method according to any one of claims 1 to 3, wherein the cells are mesenchymal stem cells.

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

6. The method according to any one of claims 1 to 5, wherein the cells are frozen cells.

7. The method according to any one of claims 1 to 6, wherein the culturing is carried out until the cells reach a confluent state.

8. The culture is 500 cm 2 The method according to any one of claims 1 to 7, which is carried out in a culture vessel having a bottom area of ​​at least 1000 mm.

9. The method according to any one of claims 1 to 8, wherein the culture substrate is a laminin fragment having integrin binding activity.

10. The method according to any one of claims 1 to 8, wherein the culture substrate is a modified laminin fragment having integrin binding activity.

11. A method for producing a cell product, comprising: culturing cells in a growth medium according to the method of any one of claims 1 to 10 to obtain grown cells in an adherent state; and culturing the grown cells in a production medium while maintaining the adherent state, to cause the cells to produce a cell product.

12. A method for producing a cell product, comprising: culturing cells in a growth culture medium according to the method of any one of claims 1 to 10 to obtain grown cells in an adherent state; culturing the grown cells in a production culture medium while maintaining the adherent state, thereby causing the cells to produce a cell product; and, after culturing in the production culture medium, culturing the grown cells in a recovery culture medium while maintaining the adherent state, wherein the culturing in the production culture medium and the culturing in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells.

13. The method of claim 11 or 12, wherein the production culture medium is a culture medium that does not contain any xenogeneic components.

14. The method according to any one of claims 11 to 13, wherein the production culture medium is a culture medium that does not contain cytokines or insulin.

15. The method according to any one of claims 11 to 14, wherein the production culture medium is a protein-free culture medium.

16. The method of any one of claims 11 to 15, further comprising recovering the supernatant of the production broth after said culturing in said production broth.

17. A method for producing a stem cell culture supernatant, comprising: culturing stem cells in a growth culture medium by adhesion culture in the presence of a culture substrate selected from laminin fragments and modified forms thereof having integrin binding activity to obtain proliferated cells in an adherent state; culturing the proliferated cells in a production culture medium while maintaining the adherent state to cause the cells to produce a cell product; and after the culture in the production culture medium, culturing the proliferated cells in a recovery culture medium while maintaining the adherent state, wherein the culture in the production culture medium and the culture in the recovery culture medium are alternately repeated while maintaining the adherent state of the cells, and the method further comprises recovering the supernatant of the production culture medium after the culture in the production culture medium.

18. The method according to claim 17, wherein the supernatant of the production culture medium is collected after the second or subsequent culture in the production culture medium.

19. The method of claim 17 or 18, wherein the stem cells are mesenchymal stem cells.

20. A method for producing a mesenchymal stem cell population having a reduced HLA-ABC positivity rate, comprising culturing mesenchymal stem cells in a growth medium in the presence of a culture substrate selected from laminin fragments and variants thereof having integrin binding activity, to obtain proliferated cells.

21. A mesenchymal stem cell population in which the ratio of HLA-ABC positive mesenchymal stem cells is 70% or less.

22. The mesenchymal stem cell population according to claim 21, wherein the proportion of CD105-positive mesenchymal stem cells is 50% or less.

23. A stem cell culture supernatant containing 5000 pg / mL or more of HGF.

24. A stem cell culture supernatant containing 50 pg / mL or more of a CD9 / CD63 EC domain fusion protein.

25. The culture supernatant according to claim 23 or 24, further containing 3000 pg / mL or more of MCP-1, 1000 pg / mL or more of GRO, and 5 μg / mL or more of fibronectin.

26. The culture supernatant according to any one of claims 23 to 25, further containing 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.

27. The culture supernatant according to any one of claims 23 to 26, which does not contain at least one of insulin, transferrin, and albumin.

28. The culture supernatant according to any one of claims 23 to 27, which does not contain recombinant proteins.

29. The culture supernatant according to any one of claims 23 to 28, which contains at least one of IL-1α, IL-1β, and TNF-α in an amount of 0 to 15 pg / mL.

30. The culture supernatant according to any one of claims 23 to 29, which contains 20 or more cytokines, each of which has a concentration of 10 pg / mL or more.

31. The culture supernatant according to any one of claims 23 to 30, wherein the culture supernatant is a culture supernatant of mesenchymal stem cells.

32. The culture supernatant according to any one of claims 23 to 31, wherein the culture supernatant is a culture supernatant of umbilical cord-derived mesenchymal stem cells.

33. A therapeutic agent comprising the mesenchymal stem cell population according to claim 21 or 22 or the culture supernatant according to any one of claims 23 to 32.