Pharmaceutical composition, method for producing three-dimensional culture of mesenchymal stem cells, method for producing exosomes, and method for producing pharmaceutical composition

Abstract

The purpose of the present invention is to provide a novel pharmaceutical composition that is efficacious for protecting or improving cognitive function. A pharmaceutical composition that contains as an active ingredient at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a and miR-369; a three-dimensional culture of mesenchymal stem cells in which the expression amount of at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a and miR-369 is increased; or a method for producing a three-dimensional culture, said method comprising a culture step for three-dimensionally culturing mesenchymal stem cells using a culture carrier containing a hydrogel and a collection step for collecting the three-dimensional culture by separating the same from the culture carrier.

Description

Pharmaceutical composition, method for producing three-dimensional culture of mesenchymal stem cells, method for producing exosomes, and method for producing pharmaceutical composition

[0001] The present invention relates to a pharmaceutical composition, a method for producing a three-dimensional culture of mesenchymal stem cells, a method for producing exosomes, and a method for producing a pharmaceutical composition.

[0002] It is estimated that there are approximately 4.62 million dementia patients in Japan. Alzheimer's dementia has the largest number of patients among all dementia types. Alzheimer's disease (AD) is believed to be caused by denatured proteins (Aβ or tau) in the brain. Under these circumstances, in order to prevent, improve, or ameliorate cognitive function by suppressing the accumulation of denatured proteins in the brain, for example, Patent Document 1 discloses antibody therapy against Aβ, Patent Document 2 discloses a drug that suppresses Aβ production, and Patent Document 3 discloses an anti-tau antibody. Clinical trials are being conducted to verify the effectiveness of treatment methods using these.

[0003] Japanese Patent Publication No. 2014-037413 Japanese Patent Publication No. 2020-083815 Japanese Patent Publication No. 2020-505056

[0004] However, the above-mentioned conventional techniques have not demonstrated efficacy in preventing, enhancing, or improving cognitive function. Therefore, the development of a new treatment method has been an issue. One aspect of the present invention aims to provide a novel pharmaceutical composition that is effective in preventing or improving cognitive function.

[0005] In order to solve the above problems, one aspect of the present invention provides a pharmaceutical composition for preventing or ameliorating cognitive decline, which comprises at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369 as an active ingredient.

[0006] In order to solve the above problems, another aspect of the present invention provides a pharmaceutical composition for preventing or ameliorating cognitive decline, characterized in that it comprises, as an active ingredient, an expression vector comprising a polynucleotide comprising a base sequence encoding let-7a, miR-26a, miR-27b, miR-34a, and miR-369.

[0007] In order to solve the above-mentioned problems, another aspect of the present invention provides a pharmaceutical composition for preventing or ameliorating cognitive decline, comprising a three-dimensional culture of mesenchymal stem cells as an active ingredient, wherein the three-dimensional culture of mesenchymal stem cells is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to a two-dimensional culture of mesenchymal stem cells.

[0008] In order to solve the above-mentioned problems, another aspect of the present invention provides a pharmaceutical composition for preventing or ameliorating cognitive decline, comprising exosomes as active ingredients containing at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369, wherein the exosomes are derived from a three-dimensional culture of mesenchymal stem cells, and the content of at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is increased compared to exosomes secreted from a two-dimensional culture of the mesenchymal stem cells.

[0009] In order to solve the above-mentioned problems, according to another aspect of the present invention, there is provided a method for producing a three-dimensional culture of mesenchymal stem cells, comprising: a culture step of three-dimensionally culturing the mesenchymal stem cells using a culture carrier comprising a hydrogel whose main component is a polysaccharide polymer, or a culture carrier comprising collagen as a main component; and a recovery step of separating the three-dimensional culture obtained in the culture step from the culture carrier and recovering the three-dimensional culture, wherein the three-dimensional culture is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to a two-dimensional culture of the mesenchymal stem cells.

[0010] In order to solve the above-mentioned problems, according to another aspect of the present invention, there is provided a method for producing a three-dimensional culture of mesenchymal stem cells, the method comprising: a culturing step of culturing spheroids as a three-dimensional culture using a spheroid culture plate; and a recovering step of recovering the three-dimensional culture obtained in the culturing step from the spheroid culture plate, wherein the three-dimensional culture is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells.

[0011] According to one aspect of the present invention, a novel pharmaceutical composition that is effective in preventing or improving cognitive function can be provided.

[0012] This is a plot diagram showing the expression levels of microRNAs in the hippocampus of subjects with Alzheimer's pathology in a group without dementia (AD-N) and a group with dementia (AD-D). The vertical axis represents microRNA expression levels in AD-N, and the horizontal axis represents microRNA expression levels in AD-D. In the plot diagram, the plot labeled 1 represents miR-26a, the plot labeled 2 represents let-7a, the plot labeled 3 represents miR-27b, the plot labeled 4 represents miR-34a, and the plot labeled 5 represents miR-369. This diagram shows the expression levels of PTEN, EGFR, BRCA1, and ESR1 in N-N (no Alzheimer's pathology - no dementia), AD-N, and AD-D. This diagram shows an outline of an experiment to confirm the effect of microRNA administration on cognitive function in APP / PS1 mice. This diagram shows an outline of a Y-maze test. This figure shows the results of a Y-maze test in wild-type (WT) mice and APP / PS1 mice administered with a negative control mimic, a miR-26a mimic, a let-7a microRNA mimic, and a miR-26a mimic plus let-7a microRNA mimic. This figure shows the morphology of 2D BM-MSCs. The scale bar indicates 100 μm. This figure shows the morphology of 3D BM-MSCs cultured using Vitrogel as a culture carrier. The scale bar indicates 100 μm. This figure shows the results of measuring the expression levels of OCT4, SOX2, and NANOG in each BM-MSC. This figure shows the results of measuring the expression levels of miR-26a and let-7a in exosomes isolated from the culture medium of 2D BM-MSCs and 3D BM-MSCs.

[0033] Figure 1 shows an outline of an experiment to confirm the effect of BM-MSC administration on the cognitive function of APP / PS1 mice. Figure 2 shows the results of a Y-maze test on APP / PS1 mice that were intrathecally administered with 2D BM-MSC. Figure 3 shows the results of a Morris water maze test (Hidden platform test) on APP / PS1 mice that were intrathecally administered with 2D BM-MSC. Figure 4 shows the results of a Morris water maze test (Probe test) on APP / PS1 mice that were intrathecally administered with 2D BM-MSC.

[0033] Figure 1 shows the results of a Y-maze test of APP / PS1 mice to which 3D BM-MSCs were intrathecally administered. Figure 2 shows the results of a Morris water maze test (Hidden platform test) of APP / PS1 mice to which 3D BM-MSCs were intrathecally administered. Figure 3 shows the results of a Morris water maze test (Probe test) of APP / PS1 mice to which 3D BM-MSCs were intrathecally administered. Figure 4 shows image data of the cisterna magna of mice 1 day, 4 days, and 28 days after intrathecal administration of PKH-labeled 3D BM-MSCs. Figure 5 shows the results of optical microscopic observation of AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using a collagen membrane. Figure 6 shows the results of electron microscopic observation of AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using a collagen membrane.

[0033] Figure 1 shows the results of measuring OCT4 expression levels, SOX2 expression levels, and NANOG expression levels in AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using a collagen membrane. Figure 2 shows the results of measuring miR-26a expression levels, let-7a expression levels, miR-27b expression levels, miR-34a expression levels, and miR-369 expression levels in exosomes isolated from the culture medium of AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using a collagen membrane. Figure 3 shows an outline of an experiment to confirm the effect on cognitive function of APP / PS1 mice of intranasal administration of exosomes isolated from the culture medium of AD-MSCs cultured using a collagen membrane. Figure 4 shows the results of a Y-maze test in APP / PS1 mice intranasally administered with a vehicle and in APP / PS1 mice intranasally administered with exosomes isolated from AD-MSCs cultured using a collagen membrane. 1 shows the results of observing AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using Prime Surface under an optical microscope. 2 shows the results of measuring the expression levels of OCT4, SOX2, and NANOG in AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using Prime Surface.

[0033] Figure 1 shows the results of measuring the expression levels of miR-26a, let-7a, miR-27b, miR-34a, and miR-369 in exosomes isolated from the culture medium of AD-MSCs cultured using a two-dimensional culture carrier and AD-MSCs cultured using Prime Surface. Figure 2 shows an outline of an experiment to confirm the effect on cognitive function of 5xFAD mice of intranasal administration of exosomes isolated from the culture medium of AD-MSCs cultured using Prime Surface. Figure 3 shows the results of a Y-maze test of 5xFAD mice intranasally administered with vehicle and 5xFAD mice intranasally administered with exosomes isolated from AD-MSCs cultured using Prime Surface.

[0033] Figure 1 shows an outline of an experiment to confirm the effect on cognitive function of 5xFAD mice of intranasal administration of exosomes isolated from the culture medium of AD-MSCs cultured using a two-dimensional culture carrier. Figure 2 shows the results of a Y-maze test for 5xFAD mice intranasally administered with a vehicle and 5xFAD mice intranasally administered with exosomes isolated from AD-MSCs cultured using Prime Surface. Figure 3 shows an outline of an experiment to confirm the effect on cognitive function of APP / PS1 mice of intranasal administration of exosomes obtained from AD-MSCs transfected with each microRNA mimic. This figure shows the results of a Y-maze test in APP / PS1 mice that were intranasally administered exosomes isolated from AD-MSCs transfected with a negative control miRNA mimic, and in APP / PS1 mice that were intranasally administered exosomes isolated from AD-MSCs transfected with each microRNA mimic.

[0013] Unless otherwise specified in this specification, the expression "A to B" representing a numerical range means "A or more (including and greater than A) and B or less (including and less than B)."

[0014] [Pharmaceutical Composition] Hereinafter, a pharmaceutical composition according to one embodiment of the present invention will be described in detail.

[0015] In this embodiment, a "pharmaceutical composition" refers to any composition containing an active ingredient for preventing or ameliorating cognitive decline in a subject. The ingredients contained in addition to the active ingredient are not particularly limited, and may include any additives (fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other excipients, as well as diluents). The dosage form of the pharmaceutical composition is not particularly limited, and may be, for example, a solid or liquid formulation. Hereinafter, in this embodiment and the like, even when simply referred to as a "pharmaceutical composition," it refers to a pharmaceutical composition for preventing or ameliorating cognitive decline.

[0016] As used herein, the term "subject" refers to an individual animal, preferably a mammalian individual, for example, a primate such as a human or chimpanzee, a rodent such as a mouse, rat, guinea pig or hamster, an even-toed ungulate such as a cow, goat, sheep or pig, an odd-toed ungulate such as a horse, a rabbit, a dog or a cat, or the like, and more preferably a human individual.

[0017] As used herein, "cognitive function" refers to intellectual functions such as memory ability, language ability, judgment ability, calculation ability, and executive ability. A decline in cognitive function refers to a decline in these functions compared to a healthy state. The cause of the decline in cognitive function is mainly caused by a decline in brain function, and is not particularly limited, but can be caused by, for example, lesions in the brain, changes in gene expression levels, changes in protein expression levels, etc. The decline in cognitive function due to Alzheimer's dementia caused by the accumulation of Aβ and tau, etc., is also included in "decline in cognitive function."

[0018] <Active ingredient of pharmaceutical composition> (microRNA) A pharmaceutical composition according to one embodiment of the present invention comprises at least one microRNA selected from the group consisting of hsa-let-7a-5p (hereinafter referred to as let-7a), hsa-miR-26a-5p (hereinafter referred to as miR-26a), hsa-miR-27b-3p (hereinafter referred to as miR-27b), hsa-miR-34a-5p (hereinafter referred to as miR-34a), and hsa-miR-369-3p (hereinafter referred to as miR-369) as an active ingredient. The pharmaceutical composition preferably comprises at least one microRNA selected from the group consisting of let-7a and miR-26a as an active ingredient. Let-7a, miR-26a, miR-27b, miR-34a, and miR-369 are microRNAs that have been newly discovered to have the effect of preventing or ameliorating cognitive decline through analysis of the hippocampus of subjects who had normal cognitive function despite having Alzheimer's disease pathology, typically characterized by accumulation of Aβ and tau in the brain. Pharmaceutical compositions containing these microRNAs as active ingredients can prevent or ameliorate cognitive decline in subjects. Let-7a, miR-26a, miR-27b, miR-34a, and miR-369 have at least the base sequences shown in Table 1 below.

[0019] The base sequences shown in Table 1 may have base substitutions within a range that allows the effects of the microRNA according to this embodiment to be obtained. When substitutions are made by single-base substitution, the number of substituted bases may be one or more, two or more, or three or more. The number of substituted bases may be five or less, four or less, or three or less. The position of the base substitution in the microRNA sequence is not particularly limited, and for example, the base may be substituted at at least one of the 3' end and 5' end of the microRNA sequence shown in Table 1.

[0020] Furthermore, any number of base sequences may be added to at least one of the 3'-end and 5'-end of the base sequence, as long as the effect of the microRNA according to this embodiment is obtained. For example, any base sequence may be added to enhance the stability of the microRNA. The number of added bases may be 1 or more, 3 or more, or 5 or more. Furthermore, the number of added bases may be 7 or less, 5 or less, or 3 or less.

[0021] MicroRNA may be either natural or non-natural microRNA. Natural microRNA refers to microRNA obtained from a subject's body fluids, tissues, etc. Artificial microRNA refers to microRNA produced by a conventionally known method, and may be microRNA that has been subjected to any chemical modification.

[0022] To improve the stability of microRNA in the subject's body, any residue in the microRNA may be substituted with a known residue other than adenosine, uridine, guanosine, and cytidine. For example, at least some uridine residues in the microRNA may be substituted with pseudouridine residues. This prevents the microRNA from being eliminated by the subject's immune system in the subject's body, improving the stability of the microRNA. As a result, the microRNA can stably exert its effect of preventing or improving cognitive decline, and this effect lasts for a long time.

[0023] (Vector) A pharmaceutical composition according to a further embodiment of the present invention comprises, as an active ingredient, an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding at least one of the aforementioned let-7a, miR-26a, miR-27b, miR-34a, and miR-369. The expression vector preferably comprises a polynucleotide comprising a nucleotide sequence encoding at least one of let-7a and miR-26a. Any base vector capable of expression in the subject's body can be used as the expression vector. For example, a plasmid, a phage, etc. can be used. More specifically, vectors such as phage vectors, plasmid vectors, viral vectors, and retroviral vectors, as well as cosmids and phagemids derived from combinations thereof, etc., can be used. The expression vector may be selected appropriately depending on the application, for example, depending on the target tissue.

[0024] The base sequence contained in the expression vector may contain any sequence for the purpose of obtaining the function of the expression vector, improving the function, etc. For example, the expression vector according to this embodiment may contain sequences encoding a promoter, an enhancer, etc.

[0025] The pharmaceutical composition contains the above-described expression vector as an active ingredient, and thus can express at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 in a subject to which the pharmaceutical composition is applied.

[0026] (Three-Dimensional Culture) According to a further embodiment of the present invention, a pharmaceutical composition comprises a three-dimensional culture of mesenchymal stem cells as an active ingredient. The three-dimensional culture of mesenchymal stem cells may be a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells. The three-dimensional culture is preferably a culture in which the expression level of at least one of microRNAs, let-7a and miR-26a, is increased compared to the two-dimensional culture of mesenchymal stem cells. The three-dimensional culture according to this embodiment is a culture obtained by three-dimensional culture under conditions similar to those in vivo using a three-dimensional culture support. The two-dimensional culture is a culture obtained by two-dimensional culture, such as by plate culture. Here, the term "culture" refers to the product obtained by culturing mesenchymal stem cells, i.e., the cultured mesenchymal stem cells themselves and the culture medium of the mesenchymal stem cells.

[0027] The expression levels of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 in three-dimensional cultures are sufficient as long as they are increased compared to two-dimensional cultures, and the degree of increase is not particularly limited. For example, the expression level of the microRNA in a three-dimensional culture may be 1.3 times or more, preferably 1.5 times or more, and particularly preferably 2 times or more, of the expression level of the microRNA in a two-dimensional culture. The increase in expression level may be determined, for example, based on a comparison of the average expression levels measured for multiple replicates.

[0028] The three-dimensional culture according to this embodiment is preferably a three-dimensional culture obtained by separating the three-dimensional culture support after three-dimensional culture. By administering only the three-dimensional culture to a subject, it is possible to prevent the administration of the three-dimensional culture support, which is an impurity, into the subject. Furthermore, by administering only the three-dimensional culture to a subject, it is easy to adjust the concentration of the active ingredient. For example, a pharmaceutical composition containing a high concentration of the active ingredient can be prepared.

[0029] The mesenchymal stem cells contained in the three-dimensional culture according to this embodiment secrete exosomes. In particular, the mesenchymal stem cells contained in the three-dimensional culture according to this embodiment secrete exosomes containing at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, at a higher concentration than mesenchymal stem cells contained in a two-dimensional culture prepared using the same mesenchymal stem cells. When the three-dimensional culture according to this embodiment is administered to a subject, exosomes are secreted in the target tissue of the subject, and the let-7a, miR-26a, miR-27b, miR-34a, and miR-369 contained in the exosomes have the effect of preventing or ameliorating a decline in cognitive function in the subject.

[0030] Furthermore, the three-dimensional culture preferably settles in the target tissue of a subject after administration as a pharmaceutical composition, thereby allowing exosome secretion from the three-dimensional culture to be maintained in the subject for a long period of time. For example, exosomes may be present for the target period even one month after administration.

[0031] The culture medium contained in the three-dimensional culture may be concentrated and administered to a subject. This is preferable because it increases the concentration of the active ingredient contained in the culture medium. The active ingredient contained in the culture medium may be exosomes.

[0032] The three-dimensional culture may include a spheroid. In the present disclosure, a spheroid refers to a cell mass in which cells are aggregated. Preferably, the spheroid has an approximately spherical shape.

[0033] Mesenchymal stem cells are stem cells with pluripotency and self-renewal capabilities, and are capable of differentiating not only into mesenchymal cells such as osteoblasts, chondrocytes, adipocytes, and muscle cells, but also of differentiating beyond the germ layer into cells such as nerve cells or hepatocytes. In this embodiment, mesenchymal stem cells include, but are not limited to, mesenchymal stem cells isolated or extracted from any subject and mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells. Hereinafter, mesenchymal stem cells may be referred to as MSCs.

[0034] "MSCs isolated or extracted from any subject" refers to, for example, MSCs isolated from a healthy subject or a diseased subject, and MSCs obtained from an extract from a mammalian fetal appendage.

[0035] From the viewpoint of safety when applied to a subject as an active ingredient of a pharmaceutical composition, "MSCs isolated from a healthy subject or a subject with a disease" are preferably collected from the subject to which the pharmaceutical composition is to be applied, or from another individual of the same or a closely related species as the subject. In this embodiment, the mesenchymal stem cells may be autologous cells derived from the subject, or allogeneic cells derived from another subject of the same species. For example, when the pharmaceutical composition of this embodiment is administered to a human individual, MSCs collected from a human of the same species are preferred, and cells collected from the same human individual to be administered, i.e., autologous MSCs, are particularly preferred.

[0036] MSCs may be collected from any sample, such as bone marrow fluid, adipose tissue, or dental pulp of a subject. In particular, MSCs derived from bone marrow fluid or adipose tissue, i.e., bone-marrow derived mesenchymal stem cells (BM-MSCs) or adipose derived mesenchymal stem cells (AD-MSCs), are preferred. The method for isolating MSCs from a sample may be performed according to known techniques and is not particularly limited. When bone marrow fluid is used as a sample, MSCs can be isolated by known techniques such as density gradient centrifugation or bone marrow seeding.

[0037] The "extract from a mammalian fetal appendage" may be a fetal appendage delivered from a mammal, preferably a human, as an afterbirth after the delivery of the fetus, or removed from the mother by Caesarean section. In particular, an extract obtained by preparing umbilical cord tissue, placental tissue, or fetal membrane by any method is preferred. It is particularly preferred that the extract does not contain cells with proliferative capacity derived from the donor mammal.

[0038] Mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells can be obtained by inducing differentiation from pluripotent stem cells such as induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic tumor cells (EC cells), and embryonic germ stem cells (EG cells).

[0039] (Exosomes) A ​​pharmaceutical composition according to a further embodiment of the present invention may comprise, as an active ingredient, exosomes containing at least one microRNA, including let-7a, miR-26a, miR-27b, miR-34a, and miR-369. It is particularly preferred that the exosomes comprise, as an active ingredient, exosomes containing at least one microRNA, including let-7a and miR-26a. Here, the exosomes are, for example, exosomes derived from a three-dimensional culture of mesenchymal stem cells, and have an increased content of at least one microRNA, including let-7a, miR-26a, miR-27b, miR-34a, and miR-369, compared to exosomes secreted from a two-dimensional culture of the mesenchymal stem cells.

[0040] Exosomes are secreted by three-dimensionally cultured stem cells in the above-described three-dimensional culture and are contained in the culture medium in the three-dimensional culture. Even when simply referring to "exosomes secreted from three-dimensional culture," it means that they are secreted from three-dimensionally cultured stem cells in the three-dimensional culture. The degree of increase in the microRNA is not particularly limited. For example, the content of at least one of the microRNAs let-7a, miR-26a, miR-27b, miR-34a, and miR-369 in exosomes secreted from three-dimensional cultures may be 1.3 times or more, preferably 1.5 times or more, and particularly preferably 2 times or more, of the corresponding microRNA content in exosomes secreted from two-dimensional cultures.

[0041] The method for producing a three-dimensional culture for isolating exosomes is not particularly limited as long as it can produce the exosomes according to the present embodiment. Furthermore, the method for isolating exosomes from the culture medium in the three-dimensional culture can be performed by a conventionally known method. For example, exosomes can be separated from the culture medium by centrifugation.

[0042] When isolating exosomes from the culture medium, the three-dimensional culture may or may not be removed from the three-dimensional culture support. Whether or not to remove the three-dimensional culture support from the three-dimensional culture may be determined depending on the type of three-dimensional culture support. For example, when a gel-like support is used as the three-dimensional culture support, the gel may be liquefied to remove the support from the three-dimensional culture, and the culture medium supernatant may be obtained by centrifugation. On the other hand, when a sheet-like support is used as the three-dimensional culture support, exosomes may be isolated from the culture supernatant without removing the support from the three-dimensional culture.

[0043] <Use> In one embodiment of the present invention, the pharmaceutical composition is a pharmaceutical composition for preventing or ameliorating cognitive decline, which may be, for example, Alzheimer's disease.

[0044] A method for treatment according to one embodiment of the present invention is a method for preventing or ameliorating cognitive decline using the pharmaceutical composition, particularly preferably a method for treating Alzheimer's disease using the pharmaceutical composition.

[0045] In the treatment method, the method of administering the pharmaceutical composition, such as the number of times the pharmaceutical composition is administered and the route of administration, is not particularly limited, and may be changed appropriately based on the condition of the subject and other medical factors.

[0046] The pharmaceutical composition may be administered to a subject via any route of administration to treat cognitive decline. The method of administering the pharmaceutical composition to a subject is not particularly limited, and may be, for example, intrathecal administration, intranasal administration, intravascular administration, etc. For example, when the active ingredient contained in the pharmaceutical composition is the microRNA, the three-dimensional culture, or the exosome, the method of administering the pharmaceutical composition is not particularly limited, and may be, for example, intrathecal administration, intranasal administration, intravascular administration, etc.

[0047] The amount of the active ingredient contained in the pharmaceutical composition is determined appropriately depending on the condition of the subject, the route of administration, and other medical factors, and is not particularly limited.

[0048] In a preferred embodiment, the dosage of the three-dimensional culture of mesenchymal stem cells is 100 mg / kg of body weight of the individual to be administered in the case of systemic administration. ４ cells ~10 ９ The cells may be 10 ５ cells ~10 ８ In the case of local administration, 10 cells per kg of body weight of the individual to be administered ２ cells ~10 ９ The cells may be 10 ４ cells ~10 ７ It is a cell.

[0049] Furthermore, other modes of administration of pharmaceutical compositions may be combined with known methods. For example, the drug delivery system used to deliver the pharmaceutical composition to the target tissue may be determined arbitrarily at the time of application of the pharmaceutical composition. Furthermore, the pharmaceutical composition may be used in combination with a known cognitive function improving agent.

[0050] [Method for Producing Three-Dimensional Cultures] One embodiment of the present invention relates to a method for producing a three-dimensional culture of mesenchymal stem cells, comprising a culturing step of three-dimensionally culturing the mesenchymal stem cells using a culture support, and a recovery step of separating the three-dimensional culture obtained in the culturing step from the culture support and recovering the three-dimensional culture. The three-dimensional culture of mesenchymal stem cells is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells. In particular, exosomes isolated from the three-dimensional culture obtained by this production method can be exosomes in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the exosomes isolated from the same two-dimensional culture of mesenchymal stem cells. The three-dimensional culture or exosomes may have increased expression levels of at least one type of microRNA, particularly let-7a and miR-26a, compared to a two-dimensional culture of the same mesenchymal stem cells or exosomes derived from the two-dimensional culture.

[0051] This method for producing a three-dimensional culture can produce a suitable three-dimensional culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased. Furthermore, a three-dimensional culture containing at least 40% or at least 50% of the number of cells seeded before culturing can be obtained.

[0052] In the method for producing a three-dimensional culture of mesenchymal stem cells, the mesenchymal stem cells are preferably bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

[0053] (Culturing Step) The culture carrier used in the culturing step is not particularly limited as long as it is a three-dimensional culture carrier that expresses at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 at a higher level than when the same two-dimensional culture carrier is used for mesenchymal stem cells.

[0054] The number of days for which the three-dimensional culture is performed may be 0.1 days or more, preferably 0.2 days or more. The number of days for which the three-dimensional culture is performed may be 7 days or less, preferably 6 days or less. When the number of days for which the three-dimensional culture is performed is within a range that satisfies the lower and upper limits, a three-dimensional culture product can be preferably obtained.

[0055] The temperature conditions for performing the three-dimensional culture may be 0° C. or higher, and preferably 4° C. or higher. The temperature conditions for performing the three-dimensional culture may be 40° C. or lower, and preferably 38° C. or lower. When the temperature conditions satisfy the upper and lower limits, a three-dimensional culture can be suitably obtained.

[0056] In one embodiment of the present invention, the culture step may be a step of three-dimensionally culturing mesenchymal stem cells using a culture carrier containing a hydrogel whose main component is a polysaccharide polymer, or a culture carrier containing collagen as its main component.

[0057] The culture process using a hydrogel is particularly preferable when administering the three-dimensional culture itself to a subject, since it can obtain a sufficient amount of high-quality cells. Furthermore, the culture carrier preferably contains, in addition to the hydrogel, a cell culture medium and a sugar solution for diluting the hydrogel.

[0058] The main component of the hydrogel of this embodiment may be a polysaccharide polymer. Here, the main component of the hydrogel contained in the culture carrier used in the culture step refers to the component that accounts for the largest amount among the components of the hydrogel. The polysaccharide polymer may be a synthetic polysaccharide polymer obtained by artificial synthesis. Furthermore, the polysaccharide polymer may be modified with any substituent, peptides, etc., from the viewpoint of enhancing the carrier function.

[0059] The hydrogel contained in the culture carrier is a polysaccharide polymer that binds to the Ca contained in the cell culture medium. ２＋ ions and Na ＋ The hydrogel forms a gel by reacting with cations such as ions. The viscosity of the hydrogel can be adjusted by adjusting the ratio of cell culture medium added to the polysaccharide polymer.

[0060] The type and concentration of sugar contained in the sugar solution for diluting the hydrogel are not particularly limited. The sugar contained in the sugar solution may be glucose, sucrose, galactose, fructose, mannose, or other sugars, and may contain these sugars alone or in combination. The concentration of the sugar solution may be 1% or more, preferably 2% or more, and more preferably 3% or more. The concentration of the sugar solution may be 10% or less, preferably 9% or less, and more preferably 8% or less. In this embodiment, the sugar solution may be one used to dilute the hydrogel.

[0061] In this embodiment, the volume of hydrogel added relative to the volume of cell culture medium may be 0.5 or more, preferably 0.6 or more, and more preferably 0.7 or more, assuming the volume of the cell culture medium is 1. Furthermore, the volume of hydrogel added relative to the volume of cell culture medium may be 4 or less, preferably 2 or less, and more preferably 1.5 or less, assuming the volume of the cell culture medium is 1. By adding the hydrogel to the cell culture medium at this ratio, a culture carrier with viscosity suitable for culturing mesenchymal stem cells can be obtained.

[0062] In this embodiment, when the volume of the hydrogel is 1, the amount of sugar solution added to the hydrogel may be 1 or more, preferably 2 or more, and more preferably 2.5 or more. Also, when the volume of the hydrogel is 1, the amount of sugar solution added to the hydrogel may be 5 or less, preferably 4 or less, and more preferably 3.5 or less.

[0063] When the volume of the cell culture medium is taken as 1, the amount of sugar solution added relative to the volume of the cell culture medium may be 0.5 or more, preferably 1 or more, and more preferably 1.25 or more. When the volume of the cell culture medium is taken as 1, the amount of sugar solution added relative to the volume of the cell culture medium may be 12 or less, preferably 6 or less, and more preferably 4.5 or less. This allows the dilution ratio of the hydrogel to be within a suitable range, resulting in a culture carrier with viscosity suitable for culturing mesenchymal stem cells.

[0064] The volume of the mixture of the hydrogel and the sugar solution may be 1 or more, preferably 1.6 or more, and more preferably 1.95 or more, relative to the volume of the cell culture medium being 1. Furthermore, the volume of the gel composition, which is the total volume of the hydrogel and the sugar solution, may be 16 or less, preferably 8 or less, and more preferably 6 or less, relative to the volume of the cell culture medium being 1.

[0065] The culture carrier is not particularly limited as long as it contains the hydrogel described above. An example of such a culture carrier is Vitrogel (registered trademark) provided by TheWell Bioscience LLC.

[0066] By using Vitrogel (registered trademark), in the recovery step described below, three-dimensional cultures can be recovered under gentle conditions without the need for treatments that damage the cells, such as enzyme treatment and / or chemical treatment with any solution, thereby increasing the cell recovery rate.

[0067] In one embodiment of the present invention, the culture carrier used in the culture step is preferably a culture carrier containing collagen. A culture step using collagen can obtain a sufficient amount of high-quality cells, and is therefore particularly preferred when the three-dimensional culture itself is administered to a subject. Furthermore, the culture carrier is preferably a culture carrier that contains a cell culture medium in addition to collagen.

[0068] As the collagen cultivation support, gel-like supports, membrane-like supports, sponge-like supports, etc., are usable. In particular, membrane-like supports are preferred as the collagen cultivation support.

[0069] Examples of such collagen-containing culture carriers include the permeable collagen membrane available from KOKEN Co., Ltd. and Cellmatrix available from Nitta Gelatin Co., Ltd.

[0070] The cell culture medium is not particularly limited as long as it is capable of culturing mesenchymal stem cells, and may be a culture medium used for culturing ordinary animal cells. For example, the medium may contain fetal bovine serum, sheep serum, or the like, or may contain serum collected from the subject from which the mesenchymal stem cells are derived, or from another individual of the same or closely related species as the subject. Also, known culture media and commercially available culture media may be used. For example, commercially available complete growth media may be used. In addition, any component for culturing mesenchymal stem cells may be added to the cell culture medium. Such optional components may include, for example, physiological saline and buffer solutions.

[0071] (Recovery step) In the recovery step, the three-dimensional culture obtained in the culture step is preferably separated from the culture support by treatment with a cell separation solution and centrifugation. This configuration allows the three-dimensional culture to be recovered under mild conditions.

[0072] When a culture process is performed using a culture carrier containing a hydrogel whose main component is a polysaccharide polymer, the cell separation solution is preferably a solution that has the function of separating the culture carrier from the three-dimensional culture by removing ionized molecules in the hydrogel structure, from the viewpoint of obtaining a sufficient amount of high-quality cells. An example of such a cell separation solution is Vitrogel (registered trademark) cell recovery solution.

[0073] When a culture process is performed using a culture carrier containing collagen as a main component, the cell separation solution is preferably a solution capable of dissolving the culture carrier in order to obtain a sufficient amount of high-quality cells. Examples of such cell separation solutions include solutions containing collagenase. Alternatively, the cell separation solution may contain a protease, such as trypsin.

[0074] After treatment with the cell separation solution, the three-dimensional culture is separated from the culture carrier by centrifugation. The centrifugation conditions (time, centrifugal force, etc.) may be appropriately determined depending on the amount, concentration, viscosity, etc. of the three-dimensional culture and the culture carrier.

[0075] The centrifugal force of the centrifugation may be 100 G or more, preferably 200 G or more. The centrifugal force of the centrifugation may be 500 G or less, preferably 400 G or less. When the centrifugal force is not lower than the lower limit, the three-dimensional culture and the culture carrier can be appropriately separated. When the centrifugal force is not higher than the upper limit, a three-dimensional culture that maintains an appropriate morphology can be obtained.

[0076] The centrifugation time may be, for example, 2 minutes or more, preferably 3 minutes or more, under conditions of 300 G. The centrifugation time may be 12 minutes or less, preferably 11 minutes or less. As long as the centrifugation time is within the range satisfying the above-mentioned lower and upper limits, the three-dimensional culture and the culture carrier can be suitably separated.

[0077] The temperature condition in the recovery step may be 30° C. or higher, and preferably 35° C. or higher. The temperature condition in the recovery step may be 40° C. or lower, and preferably 38° C. or lower.

[0078] By carrying out the above-described recovery step, a culture having excellent activity can be obtained. A culture having excellent activity refers to a culture in which the expression level of at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is significantly higher than that of a culture obtained by two-dimensional culture. Furthermore, the above-described recovery step allows the three-dimensional culture to be recovered under mild conditions, making it possible to stably produce a culture having excellent activity.

[0079] [Method for Producing Spheroids] One embodiment of the present invention relates to a method for producing a three-dimensional culture of mesenchymal stem cells, comprising a culturing step of culturing spheroids as a three-dimensional culture using a spheroid culture plate, and a recovering step of recovering the three-dimensional culture obtained in the culturing step from the spheroid culture plate. The three-dimensional culture of mesenchymal stem cells is a culture in which the expression level of at least one of the microRNAs let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is increased compared to a two-dimensional culture of the same mesenchymal stem cells. In particular, exosomes isolated from the three-dimensional culture obtained by this production method can be exosomes in which the expression level of at least one of the microRNAs let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is increased compared to exosomes isolated from a two-dimensional culture of the same mesenchymal stem cells. The three-dimensional culture or exosomes may have increased expression levels of at least one type of microRNA, particularly let-7a and miR-26a, compared to a two-dimensional culture of the same mesenchymal stem cells or exosomes derived from the two-dimensional culture.

[0080] In the method for producing a three-dimensional culture of mesenchymal stem cells, the mesenchymal stem cells are preferably bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

[0081] (Spheroid Culturing Step) In one embodiment of the present invention, the culturing step may be a culturing step of culturing spheroids as three-dimensional cultures using a spheroid culture plate.

[0082] The spheroid culture plate may be, for example, a plate having a culture surface for culturing spheroids coated with a low-adhesion or non-adhesion material. The low-adhesion or non-adhesion material may be a material containing a hydrophilic polymer. Furthermore, for example, the culture surface of the spheroid culture plate may have a recess. The diameter of the opening of the recess may be 0.3 mm or more and 0.6 mm or less. The distance from the opening of the recess to the lowest surface may be 0.1 mm or more and 0.3 mm or less. The spheroid culture plate is not particularly limited, but may be a Prime Surface (registered trademark) 90 mm Petri dish provided by Sumitomo Bakelite Co., Ltd., an EZSPHERE 100 mm dish provided by AGC Technoglass, a Corning (registered trademark) CellSTACK (registered trademark) culture chamber provided by Corning, and Nunclo (trademark) Sphera (trademark) Flasks provided by Thermo Fisher Scientific.

[0083] (Spheroid Recovery Step) The three-dimensional culture can be recovered from the spheroid culture plate by any method using pipetting, centrifugation, filtration, and the like.

[0084] The temperature condition in the recovery step may be 30° C. or higher, and preferably 35° C. or higher. The temperature condition in the recovery step may be 40° C. or lower, and preferably 38° C. or lower.

[0085] By carrying out the above-described recovery step, a culture having excellent activity can be obtained. A culture having excellent activity refers to a culture in which the expression level of at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is significantly higher than that of a culture obtained by two-dimensional culture. Furthermore, the above-described recovery step allows the three-dimensional culture to be recovered under mild conditions, making it possible to stably produce a culture having excellent activity.

[0086] (Three-dimensional culture) A three-dimensional culture of mesenchymal stem cells according to one embodiment of the present invention is produced by the above-described production method. In the three-dimensional culture obtained by the above-described production method, the expression level of at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is significantly higher than in a two-dimensional culture of the same mesenchymal stem cells. In particular, in the three-dimensional culture obtained by the above-described production method, the expression level of at least one of let-7a and miR-26a is significantly higher than in a two-dimensional culture of the same mesenchymal stem cells.

[0087] The three-dimensional culture of this embodiment is described as a product by a so-called manufacturing method because it is extremely difficult to unambiguously define the structural characteristics of the three-dimensional culture itself obtained as a result of the above-mentioned stem cell culture, and because it is impossible and impractical to investigate all possible configurations in order to measure the structural characteristics before filing the application.

[0088] [Method for Producing Exosomes] A method for producing exosomes, which is one embodiment of the present invention, includes the step of producing a three-dimensional culture of mesenchymal stem cells by the above-described production method, and the step of separating exosomes from the three-dimensional culture.

[0089] In the separation step, exosomes may be separated from the three-dimensional culture by any method. For example, the separation step may include a step of obtaining a culture supernatant by centrifuging the three-dimensional culture. The separation step may also include a step of extracting or purifying exosomes from the supernatant. The step of extracting or purifying exosomes from the supernatant may include, for example, ultracentrifugation, ultrafiltration, chromatography, or treatment with a separation reagent.

[0090] The centrifugal force of the centrifugation in the process of obtaining the supernatant may be 100 G or more, preferably 200 G or more. The centrifugal force of the centrifugation may be 500 G or less, preferably 400 G or less. When the centrifugal force is not lower than the lower limit, the exosome-containing supernatant and the cultured mesenchymal stem cells can be appropriately separated. When the centrifugal force is not higher than the upper limit, the exosome-containing supernatant can be obtained with minimal damage.

[0091] The centrifugation time in the process of obtaining the supernatant may be, for example, 2 minutes or more, preferably 3 minutes or more, under conditions of 300 G. The centrifugation time may be 20 minutes or less, preferably 15 minutes or less. As long as the centrifugation time is within the range that satisfies the above-mentioned lower and upper limits, the exosome-containing supernatant and the cultured mesenchymal stem cells can be suitably separated.

[0092] The temperature condition in the separation step may be 0° C. or higher, and preferably 2° C. or higher. The temperature condition in the separation step may be 40° C. or lower, and preferably 38° C. or lower.

[0093] (Exosomes) Exosomes according to one embodiment of the present invention are produced by the above-described exosome production method. In exosomes obtained by the above-described production method, the expression level of at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is significantly higher than that of exosomes secreted from two-dimensional cultures of the same mesenchymal stem cells. In particular, in exosomes obtained by the above-described production method, the expression level of at least one of let-7a and miR-26a is significantly higher than that of exosomes secreted from two-dimensional cultures of the same mesenchymal stem cells. The exosomes may be particles measuring 40 nm or more and 400 nm or less.

[0094] The exosomes of this embodiment are described as products by a so-called manufacturing method because it is extremely difficult to unambiguously define the structure of the exosomes themselves secreted from the three-dimensional culture obtained as a result of the above-mentioned stem cell culture, and because it is impossible and impractical to investigate all possible structures in order to measure their structural characteristics before filing the application.

[0095] [Method for producing pharmaceutical composition] A method for producing a pharmaceutical composition according to one embodiment of the present invention comprises the steps of producing a three-dimensional culture of mesenchymal stem cells by the above-described production method, and producing a pharmaceutical composition for preventing or ameliorating cognitive decline using the three-dimensional culture.

[0096] Furthermore, a method for producing a pharmaceutical composition according to one embodiment of the present invention includes the steps of producing exosomes by the above-described production method, and producing a pharmaceutical composition for preventing or ameliorating cognitive decline using the exosomes.

[0097] The process for producing the pharmaceutical composition is not particularly limited, and may be applied taking into consideration the condition of the subject and other medical factors.

[0098] [Exosome Screening Method] An exosome screening method according to one embodiment of the present invention may include a step of selecting exosomes using an increase in the expression level of at least one microRNA, including let-7a, miR-26a, miR-27b, miR-34a, and miR-369, as an indicator. In particular, the exosome screening method preferably includes a step of selecting exosomes using an increase in the expression level of at least one microRNA, including let-7a and miR-26a, as an indicator. The degree of increase in expression level is not particularly limited, but, for example, the expression level of the microRNA in exosomes may be 1.3-fold or more, preferably 1.5-fold or more, and particularly preferably 2-fold or more, relative to the expression level of the microRNA in exosomes isolated from a two-dimensional culture.

[0099] Exosomes selected by the above screening method are useful as drugs for preventing or improving cognitive decline.

[0100] [Culture] A culture according to one embodiment of the present invention may be one in which the expression level of at least one microRNA, including let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to a control culture. Here, the culture may be a culture obtained by three-dimensional culture, a culture transfected with each microRNA, or the like. When the culture according to one embodiment of the present invention is a culture obtained by three-dimensional culture, the control culture is a culture obtained by two-dimensional culture. When the culture according to one embodiment of the present invention is a culture transfected with each microRNA, the control culture is an untransfected culture or a culture transfected with a negative control miRNA mimic.

[0101] [Therapeutic Method] A therapeutic method according to one embodiment of the present invention may involve administering a pharmaceutical composition containing the above-described microRNA, three-dimensional culture, and / or exosome as an active ingredient. The therapeutic method may be a therapeutic method aimed at preventing or ameliorating cognitive decline, particularly Alzheimer's disease. The pharmaceutical composition may be administered, for example, by intrathecal administration, intranasal administration, or intravascular administration.

[0102] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0103] [Summary] The present invention can also be expressed as follows.

[0104] The pharmaceutical composition according to this aspect 1 is a pharmaceutical composition for preventing or ameliorating cognitive decline, characterized by comprising at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369 as an active ingredient. This configuration makes it possible to provide a novel pharmaceutical composition intended to prevent or ameliorate cognitive decline.

[0105] A pharmaceutical composition according to Aspect 2 is the pharmaceutical composition according to Aspect 1, characterized in that at least some of the uridine residues in the microRNA are substituted with pseudouridine residues. This configuration can increase the stability of the microRNA in the subject.

[0106] The pharmaceutical composition according to Aspect 3 is a pharmaceutical composition for preventing or ameliorating cognitive decline, comprising as an active ingredient an expression vector comprising a polynucleotide comprising a base sequence encoding at least one of let-7a, miR-26a, miR-27b, miR-34a, and miR-369. This configuration enables let-7a, miR-26a, miR-27b, miR-34a, and miR-369 to be expressed in a subject.

[0107] A pharmaceutical composition according to Aspect 4 is a pharmaceutical composition for preventing or ameliorating cognitive decline, comprising a three-dimensional culture of mesenchymal stem cells as an active ingredient, wherein the three-dimensional culture of mesenchymal stem cells is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to a two-dimensional culture of mesenchymal stem cells. This configuration makes it possible to provide a pharmaceutical composition that can express exosomes containing let-7a, miR-26a, miR-27b, miR-34a, and miR-369 in a subject.

[0108] A pharmaceutical composition according to Aspect 5 is a pharmaceutical composition for preventing or ameliorating cognitive decline, comprising as an active ingredient exosomes containing at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369, wherein the exosomes are derived from a three-dimensional culture of mesenchymal stem cells, and the content of at least one microRNA selected from let-7a, miR-26a, miR-27b, miR-34a, and miR-369 is increased compared to exosomes secreted from a two-dimensional culture of the mesenchymal stem cells. This configuration makes it possible to provide a novel pharmaceutical composition intended for preventing or ameliorating cognitive decline.

[0109] The pharmaceutical composition according to Aspect 6 is the pharmaceutical composition according to Aspect 4 or 5, characterized in that the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

[0110] A pharmaceutical composition according to this aspect 7 is the pharmaceutical composition according to any one of aspects 4 to 6, wherein the three-dimensional culture comprises a spheroid.

[0111] The pharmaceutical composition according to Aspect 8 is the pharmaceutical composition according to any one of Aspects 1 to 7, wherein the cognitive decline is due to Alzheimer's disease.

[0112] A method for producing a three-dimensional culture of mesenchymal stem cells according to Aspect 9 includes a culturing step of three-dimensionally culturing the mesenchymal stem cells using a culture carrier containing a hydrogel whose main component is a polysaccharide polymer, or a culture carrier containing collagen as a main component, and a recovery step of separating the three-dimensional culture obtained in the culturing step from the culture carrier to recover the three-dimensional culture, wherein the three-dimensional culture is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to a two-dimensional culture of the mesenchymal stem cells. This configuration makes it possible to provide a three-dimensional culture for preventing or ameliorating cognitive decline.

[0113] A method for producing a three-dimensional culture of mesenchymal stem cells according to aspect 10 includes a culturing step of culturing spheroids as a three-dimensional culture using a spheroid culture plate, and a recovering step of recovering the three-dimensional culture obtained in the culturing step from the spheroid culture plate, wherein the three-dimensional culture is a culture in which the expression level of at least one of microRNAs, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells.

[0114] The method for producing a three-dimensional culture according to Aspect 11 is a method for producing a three-dimensional culture of mesenchymal stem cells in Aspect 9 or 10, characterized in that the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

[0115] A method for producing exosomes according to Aspect 12 includes the steps of producing a three-dimensional culture of mesenchymal stem cells by the production method according to any one of Aspects 9 to 11, and isolating exosomes from the three-dimensional culture.

[0116] A three-dimensional culture according to Aspect 13 is a three-dimensional culture of mesenchymal stem cells, characterized by being produced by any one of Aspects 9 to 11. A three-dimensional culture of this configuration has properties suitable for use in preventing or improving cognitive decline.

[0117] The exosomes according to Aspect 14 are exosomes produced by the production method of Aspect 12.

[0118] A method for producing a pharmaceutical composition according to Aspect 15 includes the steps of producing a three-dimensional culture of mesenchymal stem cells by the method described in Aspect 11, and producing a pharmaceutical composition for preventing or ameliorating cognitive decline using the three-dimensional culture. [Examples] Example 1: Cognitive Function of Donated Bodies with Alzheimer's Pathology Postmortem brains of subjects donated to Sapporo Medical University were examined for Alzheimer's disease (AD) pathology and cognitive function during life (Ethics Committee Approved Research: 26-2-51). Subjects were selected who had no visible lesions such as cerebral infarction or cerebral hemorrhage, and no history of cerebrovascular disease, brain diseases other than AD (such as dementia with Lewy bodies), or psychiatric disorders (such as depression).

[0119] The presence or absence of AD pathology in the subjects was evaluated as follows. First, ABC scores were calculated: 1) Phase for Aβ plaques (A score), 2) Braak NFT stage (B score), and 3) CERAD neuritic plaque score (C score). Next, according to the diagnostic criteria of the National Institute on Aging-Alzheimer's Association, AD pathology was classified as Not / Low / Intermediate / High. Not / Low was evaluated as "no AD pathology," and Intermediate / High was evaluated as "present with AD pathology."

[0120] The cognitive function of the subjects was evaluated based on the presence or absence of dementia. The presence or absence of dementia was assessed by questioning the bereaved family members about the cognitive function of the subjects 6 months before their death according to the Clinical Dementia Rating (CDR). A CDR score of 0 or 0.5 was considered "no dementia," while a CDR score of 1, 2, or 3 was considered "presence of dementia."

[0121] Here, the group without AD pathology (N) was divided into a group without dementia (N-N) and a group with dementia (N-D), and the group with AD pathology (AD) was divided into a group without dementia (AD-N) and a group with dementia (AD-D). The number of subjects classified into each group was 4 to 5.

[0122] Example 2: Analysis of microRNA expression levels MicroRNAs were extracted from the hippocampus of each group, and array analysis was performed on the total microRNA expression levels. Array analysis was performed using a microarray kit (Thermo Fisher Scientific), and data were analyzed using the ΔΔCt method. The results of the array analysis of AD-N and AD-D were compared. Figure 1 is a plot diagram showing the results. In the plot diagram of Figure 1, miR-16 is used as the reference gene, the expression level of microRNA in AD-N is shown on the vertical axis, and the expression level of microRNA in AD-D is shown on the horizontal axis, with each gene plotted based on its expression level.

[0123] Among the microRNAs that were expressed at higher levels in AD-N compared to AD-D, the expression levels of hsa-miR-26a-5p (hereinafter "miR-26a") and hsa-let-7a-5p (hereinafter "let-7a") were particularly high. In Figure 1, the plot marked 1 indicates miR-26a, and the plot marked 2 indicates the expression level of let-7a. In addition, microRNAs that were expressed at higher levels in AD-N compared to AD-D included hsa-miR-27b-3p (hereinafter "miR-27b"), hsa-miR-34a-5p (hereinafter "miR-34a"), and hsa-miR-369-3p (hereinafter "miR-369"). In FIG. 1, the plot indicated as 3 indicates the expression level of miR-27b, the plot indicated as 4 indicates the expression level of miR-34a, and the plot indicated as 5 indicates the expression level of miR-369.

[0124] Furthermore, the mRNA expression levels of PTEN, EGFR, BRCA1, and ESR1, target genes controlled by both let-7a and miR-26a, were analyzed in the three groups N-N, AD-N, and AD-D. Total RNA was extracted from samples from each group using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific). cDNA was synthesized by reverse transcription, and real-time PCR was performed on PTEN, EGFR, BRCA1, and ESR1 using primer sets corresponding to the sequence numbers shown in Table 2.

[0125] The results for the mRNA expression levels of each gene are shown in Figure 2. The mRNAs of these genes showed higher expression levels in AD-D than in AD-N. In Figure 2, the average mRNA expression levels of AD-D and AD-N are shown as relative values ​​when the average mRNA expression level of N-N is set to 1, and the error bars indicate the standard error in each group.

[0126] Example 3 MicroRNA Administration Experiment (Preparation of Mice) A negative control mimic (75 pmol), a miR-26a mimic (75 pmol), a let-7a mimic (75 pmol), and a mixture of miR-26a and let-7a mimics (75 pmol each, hereinafter referred to as "miR-26a + let-7a mimic") were each mixed with Invivofectamine (registered trademark) reagent (Thermo Fisher) to a total volume of 5 μl. This resulted in the preparation of a negative control mimic reagent (15 nmol / ml), a miR-26a mimic reagent (15 nmol / ml), a let-7a mimic reagent (15 nmol / ml), and a mixed reagent of miR-26a and let-7a mimics (15 nmol / ml each). The negative control mimic used was mirVana™ miRNA Mimic, Negative Control #1 (Thermo Fisher Scientific).

[0127] A hole was drilled in the skull of an 18-month-old APP / PS1 mouse (Charles River) and a 0.4 mm diameter cannula was inserted into the cerebral ventricle. The cannula was placed 0.4 mm posterior to bregma, 1 mm to the right of the midline, and 2.5 mm deep. 5 μL of microRNA reagent was injected into the mouse ventricle through the cannula. Injections were performed at a rate of 1 μL / min using a 0.13 mm diameter Hamilton syringe. This treatment was performed four times, one week apart, to prepare mice for the following groups: negative control mimic, miR-26a mimic, let-7a mimic, and miR-26a + let-7a mimic. Figure 3 shows a schematic diagram of this treatment. APP / PS1 mice are genetically prone to accumulate large amounts of Aβ, and already exhibited cognitive impairment at 18 months of age.

[0128] (Y-maze test) A Y-maze test was performed using a Y-maze apparatus (Muromachi Kikai Co., Ltd.) consisting of three arms, each 41.5 cm long, 4 cm wide, and 10 cm high, connected at a 120° angle. Figure 4 shows a schematic diagram of the Y-maze test. Wild-type mice and APP / PS1 mice treated with each microRNA reagent were placed at the end of one of the arms of the Y-maze apparatus and allowed to freely explore the maze for 7 minutes, with the arms they entered recorded in order. The number of times the mice entered each arm during the measurement period (total arm entries) and the number of combinations in which they entered three consecutive arms (number of alternations) were measured. The alternation rate (%) was calculated using the following formula and used as an index of short-term memory.

[0129] Alternation rate (%) = number of alternations / (total number of arm entries - 2) × 100 The measured alternation rate (Percentage of Alteration) is shown in Figure 5. As is clear from Figure 5, cognitive function was significantly improved in the miR-26a mimic administration group, the let-7a mimic administration group, and the miR-26a + let-7a mimic administration group compared to the negative control mimic administration group.

[0130] Example 4: Preparation of three-dimensional mesenchymal stem cells Mesenchymal stem cells (BM-MSCs) were obtained by culturing bone marrow leaked during total hip replacement surgery from subjects (n=4) with osteoarthritis of the hip (Sapporo Medical University Ethics Committee Approved Research 322-1170). Details of the subjects are shown in Table 3. BM-MSCs were seeded onto three-dimensional culture carriers and three-dimensional culture was performed. Vitrogel (registered trademark, TheWell Bioscience LLC) and Neoveil Nano (registered trademark, Gunze Co., Ltd.) were used as three-dimensional culture carriers.

[0131] The hydrogel included in the Vitrogel kit was serially diluted with VitroGel Dilution Solution or 5% sucrose solution to a hydrogel:sucrose solution (v / v) ratio of 1:3. The diluted hydrogel:BM-MSC suspension was mixed at a ratio of 4:1 to obtain a mixture. The final cell concentration in the mixture was 2.7 × 10 ５ The mixture was then added to a 24-well plate (Costar® 24-Well Cell Culture Cluster, Corning) and allowed to stabilize at room temperature for 15 minutes. 300 μL of complete growth medium (D-MEM High Glucose, SIGMA-ALDRICH + 10% fetal bovine serum, Cell Culture Bioscience) was then added to the mixture. The mixture was maintained at 37°C for 2 days to allow for three-dimensional culture.

[0132] 1 mL of Vitrogel® cell recovery solution (TheWell Bioscience LLC) was added to a 24-well plate where three-dimensional culture was performed, and the suspension was then mixed with 5 mL of Vitrogel cell recovery solution and placed in a 15 mL centrifuge tube (BM Equipment). The centrifuge tube was then inverted 20 times to mix and placed in a 37°C water bath (Iuchi Co., Ltd.) for 2 minutes. This mixing and placing process was repeated five times. Then, the tube was centrifuged at 300 G for 5 minutes using a centrifuge (Tomy Seiko Co., Ltd.). The precipitate obtained as a three-dimensional culture was the result of centrifugation. The number of cells obtained as a three-dimensional culture was approximately 40-60% of the cells initially seeded.

[0133] BM-MSCs were seeded onto 10 x 10 cm Neoveil Nano (a nonwoven fabric containing polyglycolic acid) and 13.4 mL of MSC culture medium was added. The BM-MSCs were maintained at 37°C for 2 days to obtain a three-dimensional culture. To separate the three-dimensional culture from the Neoveil Nano, the three-dimensional culture was treated with trypsin.

[0134] As a result of three-dimensional culture using Neoveil Nano, the number of cells obtained as a three-dimensional culture was approximately 10% of the cells initially seeded. From the perspective of obtaining three-dimensional cultures, the three-dimensional culture method using Vitrogel was found to have superior culture efficiency compared to the three-dimensional culture method using Neoveil Nano.

[0135] Example 5: Administration of cultures (Preparation of BM-MSCs) Three-dimensional cultures (3D BM-MSCs) were obtained by three-dimensional culture using Vitrogel in the same manner as in Example 4. Human-derived BM-MSCs obtained from the same subject as in Example 4 were cultured on a two-dimensional culture carrier (Costar (registered trademark) 24-Well Cell Culture Cluster, manufactured by Corning) at a concentration of 2 × 10 ４ Human-derived BM-MSCs were seeded at 100 cells / well and cultured for 2 days.

[0136] Each culture was photographed using an optical microscope (Nikon). The morphologies of 2D-MSCs and 3D-MSCs are shown in Figures 6 and 7. As shown in Figure 6, 2D-MSCs exhibited a spindle shape. On the other hand, as shown in Figure 7, 3D-MSCs exhibited a rounded morphology within the gel.

[0137] Furthermore, RT-PCR was performed to measure the mRNA expression levels of the stem cell markers OCT4, SOX2, and NANOG using primer sets corresponding to the sequence numbers shown in Table 4. Total RNA was extracted from each group of samples using Tri Reagent (Molecular Research Center, Inc.) according to its protocol. cDNA was synthesized by reverse transcription, and real-time PCR was performed on OCT4, SOX2, and NANOG using primer sets corresponding to the sequence numbers shown in Table 4. The results are shown in Figure 8. As shown in Figure 8, it was revealed that the expression levels of all stem cell markers were higher in 3D-MSCs (Vitrogel) than in 2D-MSCs (control). The mRNA expression levels of each sample shown in Figure 8 are plotted as relative values ​​when the average mRNA expression level of the control is set to 1, and error bars indicate the standard error for each group.

[0138] The supernatant was collected from each of the three 3D cultures of 3D AD-MSCs and 2D AD-MSCs derived from three cases listed in Table 3 by centrifugation at 300G for 3 minutes. The resulting supernatant was then centrifuged at 3000G for 15 minutes and collected. The supernatant was reacted with ExoQuick-TC (System Biosciences), and exosomes were isolated according to the protocol.

[0139] The miRNA contained in exosomes was isolated using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (Thermo Fisher Scientific). Furthermore, cDNA was prepared using the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). The expression level of microRNA was measured by PCR using the TaqMan Advanced miRNA Assay (Thermo Fisher Scientific). The results are shown in Figure 9. As shown in Figure 9, it was revealed that the expression levels of let-7a and miR-26a microRNAs were higher in 3D-MSCs (Vitrogel) than in 2D-MSCs (control). The expression levels of microRNAs in 3D-MSCs derived from each case shown in Figure 9 are plotted as relative values ​​when the expression level of microRNAs in 2D-MSCs of the corresponding case is set to 1.

[0140] (Administration) 2D BM-MSC (2.5 × 10 ４ cells / animal) and 3D BM-MSCs (2.5 × 10 ４ Each MSC suspension was prepared by suspending 1000 MSCs (1000 cells / mouse) in 5 μL of PBS. Under anesthesia, 20-month-old APP / PS1 mice were administered intrathecally by injecting the MSC suspension or vehicle (PBS) into the cisterna magna through an incision in the neck skin, subcutaneous tissue, and muscle to expose the dura mater of the cisterna magna. For intrathecal administration, the MSC suspension was slowly administered using a 10 μL Hamilton syringe. Two minutes after administration, the Hamilton syringe was removed, and the incision was sutured. The mice were maintained for one month after intrathecal administration before being used in a test to assess cognitive function (Figure 10).

[0141] Example 6: Test to confirm cognitive function Mice from the 2D BM-MSC group and the 3D BM-MSC group were subjected to a Y-maze test and a Morris water maze test (hidden platform test and probe test). Furthermore, as a control for each test of the 2D BM-MSC group and the 3D BM-MSC group, mice from the vehicle group were prepared in the same manner as the mice from the 2D BM-MSC group and the 3D BM-MSC group, except that only the vehicle was administered intrathecally. Four to eight mice were prepared in each group.

[0142] (Y-maze test) For the mice prepared in Example 5, a Y-maze test was performed in the same manner as in Example 3. The results are shown in Figures 11 and 14. As shown in Figure 11, there was no significant difference between the alternation rate (%) of mice in the 2D BM-MSC group and the vehicle group (P = 0.5158). On the other hand, as shown in Figure 14, the alternation rate (%) of mice in the 3D BM-MSC group was significantly higher than that of mice in the vehicle group (P < 0.05, unpaired t-test).

[0143] (Morris Water Maze Test) 1. Hidden Platform Test The Morris Water Maze test's Hidden Platform test was performed as follows. Specifically, mice were placed in a circular pool (1.2 m in diameter) filled with water (25°C) from a start position set at any position on the edge of the pool. The time it took to reach the goal platform (escape latency) was measured within a 60-second time limit. A goal platform (10 cm in diameter) was placed below the water surface of the pool, and visual cues were placed around the pool. Mice that failed to reach the goal within the 60-second time limit were allowed to remain on the platform for 15 seconds before the test was terminated. The same test, except for the change in the start position, was performed four times a day for four days. While swimming, mice memorized the location of the platform using visual cues placed around the pool. Therefore, in cognitively normal mice, repeated testing leads to a reduction in reach time through learning. Escape latency results for each group were analyzed using a two-way repeated measures analysis of variance. The results are shown in Figures 12 and 15. As shown in Figure 12, there was no significant difference in the escape latency of mice in the 2D-MSC group compared to the escape latency of mice in the vehicle group. On the other hand, as shown in Figure 15, mice in the 3D BM-MSC group showed a reduction in escape latency on days 3 and 4 (P<0.05).

[0144] 2. Probe Test The probe test was performed as follows. That is, on the fifth day after the four-day hidden platform test, mice were allowed to swim for 60 seconds under the same conditions as in the hidden platform test, except that the platform was removed. The number of times the mice crossed the original platform location (crossing times of the platform) was measured. This allowed us to assess the mice's ability to approach the goal based on their spatial memory. Crossing times of the platform were assessed by unpaired t-test. The results are shown in Figures 13 and 16. As shown in Figure 13, the crossing times of the 2D-MSC group mice were not significantly different from those of the vehicle group mice (p = 0.9054).On the other hand, as shown in Figure 16, the crossing times of the 3D BM-MSC group mice were significantly higher than those of the vehicle group mice (p < 0.01).

[0145] (Discussion) From the above, it was found that intrathecal administration of 3D BM-MSCs improved the cognitive function of APP / PS1 mice.

[0146] Example 7: 3D BM-MSCs in the cisterna magna 3D BM-MSCs were labeled with PKH and administered intrathecally (2.5 x 10 ４ The cisterna magna (CM) was collected from the mice 1, 14, and 28 days after intrathecal administration. 20-μm-thick frozen sections of the cisterna magna were prepared and stained with DAPI. Image data were acquired using a confocal microscope (Nikon).

[0147] Image data is shown in Figure 17. The arrows in Figure 17 indicate the locations where PKH signals were detected. In particular, the presence of PKH-labeled 3D BM-MSCs was confirmed in the subarachnoid space of the cisterna magna (CM) and the choroid plexus (CP). Furthermore, as shown in Figure 17, even 28 days after administration of 3D BM-MSCs, PKH-labeled 3D BM-MSCs were present in the cisterna magna and choroid plexus. This demonstrated that 3D BM-MSCs had established themselves in the target tissues of the administration subjects.

[0148] Example 8: Morphological evaluation of adipose-derived mesenchymal stem cells cultured three-dimensionally using a collagen membrane Three-dimensional culture and two-dimensional culture were carried out using human AD-MSCs (Lonza) derived from a 33-year-old woman with a case of caucasoid as adipose-derived mesenchymal stem cells (hereinafter referred to as "AD-MSCs").

[0149] AD-MSCs were cultured using a permeable collagen membrane CM-6 for 6-well plates (KOKEN) or a regular 2D dish equipped with a two-dimensional culture support (cell culture plate 6-well flat bottom, BM Equipment Co., Ltd.).

[0150] Two ml of medium was added to each well of a permeable collagen membrane 6-well plate (hereinafter referred to as "collagen membrane plate"), and 1 ml of medium was added to the collagen membrane, which was then immersed for at least 5 minutes to neutralize the collagen membrane. The medium used for neutralization was then removed, and 2 ml of fresh medium was added to each well to prepare a collagen membrane plate. 1 ml of each AD-MSC cell suspension (4.1 x 10 cells) was placed on the collagen membrane of the prepared collagen membrane plate. ４ After the addition, the AD-MSCs were cultured at 37°C for 2 days. ４ Each AD-MSC was seeded at 100 cells / well and cultured at 37°C for 2 days. The cultured cells were observed under an optical microscope (Nikon). The results are shown in Figure 18. As shown in Figure 18, the AD-MSCs cultured using the collagen membrane exhibited a spindle shape.

[0151] AD-MSCs cultured on collagen membrane plates and AD-MSCs cultured on two-dimensional culture supports were observed using a scanning electron microscope (Hitachi Corporation). The results are shown in Figure 19. As shown in Figure 19, AD-MSCs cultured on two-dimensional culture supports (normal 2D dishes) showed a morphology that adhered to the dish, while AD-MSCs cultured on collagen membrane plates were bulging and had many fine protrusions.

[0152] Furthermore, three-dimensional culture of human AD-MSCs (Lonza) derived from three cases was performed using collagen membrane plates. The age, sex, and race of each case from which the AD-MSCs were derived are shown in Table 5 below.

[0153] RT-PCR was performed using the same method as in Example 5 to measure the mRNA expression levels of stem cell markers OCT4, SOX2, and NANOG for AD-MSCs cultured on collagen membrane plates and AD-MSCs cultured on two-dimensional culture supports. The results are shown in Figure 20. As shown in Figure 20, the expression levels of OCT4 and NANOG tended to be higher in 3D AD-MSCs (collagen) than in 2D AD-MSCs (2D) cultured on two-dimensional culture supports. The mRNA expression levels of each sample shown in Figure 20 are plotted as relative values ​​when the average mRNA expression level of 2D-MSCs (control) is set to 1, and error bars indicate the standard deviation for each group.

[0154] The culture medium of AD-MSCs cultured on collagen membrane plates and the culture medium of AD-MSCs cultured on two-dimensional culture carriers were each centrifuged at 300G for 3 minutes to obtain supernatants. The resulting supernatants were further centrifuged at 3000G for 15 minutes, and the supernatants were collected. The supernatants were reacted with ExoQuick-TC (System Biosciences), and exosomes were isolated according to the protocol. The expression levels of microRNAs let-7a, miR-26a, miR-27b, miR-34a, and miR-369 in exosomes were measured using the same method as in Example 5. The results are shown in Figure 21. As shown in Figure 21, it was revealed that the expression levels of microRNAs miR-26a, miR-27b, and miR-369 were elevated in AD-MSCs (collagen) cultured on a collagen membrane compared to AD-MSCs (2D) cultured on a two-dimensional culture carrier. The expression levels of microRNAs in AD-MSCs (collagen) cultured on a collagen membrane derived from each case shown in Figure 21 are plotted as relative values ​​when the expression level of microRNAs in 2D-MSCs (2D) derived from the corresponding case is set to 1.

[0155] Example 9: Administration experiment of exosomes derived from adipose-derived mesenchymal stem cells cultured three-dimensionally using a collagen membrane plate. AD-MSCs derived from case 2 (37 years old, female, hispanic) in Table 5 were cultured three-dimensionally using a collagen membrane plate. The three-dimensional culture was performed in the same manner as in Example 8.

[0156] (Y-maze test) Vehicle (PBS) and exosome suspension were prepared. Here, the AD-MSC culture medium was centrifuged to obtain a supernatant, which was then further subjected to ultracentrifugation to separate exosomes. Centrifugation to obtain the supernatant from the AD-MSC culture medium was performed at 2,000 g for 10 minutes. Further ultracentrifugation to obtain a supernatant containing exosomes from the supernatant was performed: a first centrifugation at 10,000 g for 60 minutes, a second centrifugation at 100,000 g for 70 minutes, and a third centrifugation by density gradient method at 100,000 g for 16 hours. The protein content of the exosome-containing supernatant obtained as a result of ultracentrifugation was measured using the BCA method (BCA Assay and Lowry Assays, Thermo Fisher Scientific). Exosome suspensions were prepared by suspending exosomes (5 μg protein equivalent / animal) derived from AD-MSCs cultured on collagen membrane plates in 25 μl of PBS. Hyaluronidase suspensions were also prepared by suspending 100 U of hyaluronidase (Sigma-Aldrich) in 5 μl of PBS. Under anesthesia, 21-month-old APP / PS1 mice were intranasally administered 5 μl of hyaluronidase suspension. Starting 30 minutes after administration of the hyaluronidase suspension, 5 μl of exosome suspension or vehicle was administered intranasally five times, every 5 minutes, for a total of 25 μl. Similarly, hyaluronidase suspension, exosome suspension, or vehicle were intranasally administered four times, one week apart. One month after the initial administration, the mice were tested for cognitive function (Figure 22).

[0157] A Y-maze test was performed on mice administered the exosome suspension or vehicle. The Y-maze test was performed in the same manner as in Example 3. The results are shown in Figure 23. As shown in Figure 23, the alternation behavior rate (%) of mice in the group administered exosomes derived from AD-MSCs cultured on collagen membrane plates was significantly higher than the alternation behavior rate (%) of mice in the vehicle group (P<0.05, unpaired t-test).

[0158] Example 10: Morphological evaluation of spheroid-cultured adipose-derived mesenchymal stem cells Three-dimensional and two-dimensional cultures were performed using human AD-MSCs (Lonza) derived from a 33-year-old woman with a case of caucasoid disease. For three-dimensional culture, Prime Surface 90 mm petri dishes (Sumitomo Bakelite Co., Ltd.) were used. 9 ml (4.0 × 10) of AD-MSC cell suspension was placed in the petri dish. ５ For two-dimensional culture, 9 ml of the AD-MSC cell suspension (4.0 × 10 cells / min) was seeded on a two-dimensional culture support (100 mm cell culture dish, manufactured by BM Equipment Co., Ltd.). ５ The cells were then cultured at 37°C for 2 days, and the resulting culture was observed under an optical microscope (Nikon). The results are shown in Figure 24. When the Prime Surface Petri dish was used, AD-MSCs formed spheroids.

[0159] Furthermore, RT-PCR was performed using the same method as in Example 5 to measure the mRNA expression levels of the stem cell markers OCT4, SOX2, and NANOG for AD-MSCs and AD-MSC spheroids cultured using two-dimensional culture supports. The results are shown in Figure 25. As shown in Figure 25, it was revealed that the expression level of NANOG in AD-MSC spheroids (Prime Surface) was elevated compared to 2D-MSCs (2D). The mRNA expression levels of each sample shown in Figure 25 are plotted as relative values ​​when the average mRNA expression level of 2D-MSCs (2D) is set to 1, and error bars indicate the standard deviation in each group.

[0160] The supernatant of the culture medium of AD-MSCs cultured on a two-dimensional culture carrier and the supernatant of the culture medium of AD-MSCs cultured on Prime Surface were collected by the same method as in Example 8. Furthermore, exosomes were isolated from each supernatant by the same method as in Example 8. For the exosome-containing supernatant, the expression levels of microRNAs let-7a, miR-26a, miR-27b, miR-34a, and miR-369 were measured by the same method as in Example 5. The results are shown in Figure 26. As shown in Figure 26, it was revealed that the expression levels of microRNAs let-7a, miR-26a, miR-34a, and miR-369 were elevated in AD-MSCs (Prime Surface) cultured on Prime Surface dishes compared to AD-MSCs (2D) cultured on a two-dimensional culture carrier. The expression levels of microRNAs in 3D-MSCs (Prime Surface) cultured using Prime Surface derived from each case shown in Figure 26 are plotted as relative values ​​when the expression level of microRNAs in 2D-MSCs (2D) of the corresponding case is set to 1.

[0161] Example 11: Administration experiment of exosomes derived from spheroid-cultured adipose-derived mesenchymal stem cells Human AD-MSCs (Lonza) derived from a 33-year-old woman with caucasoid disease were cultured as spheroids using the same method as in Example 10. (Y-maze test) Vehicle (PBS) and an exosome suspension were prepared. Here, the AD-MSC culture medium was centrifuged to obtain a supernatant, which was then concentrated using an Amicon Ultra centrifugal ultrafiltration filter (Merck). Exosomes were isolated from the concentrated supernatant using the MagCapture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Corporation). The protein content of the isolated exosomes was measured using the BCA method (BCA Assay and Lowry Assays, Thermo Fisher Scientific). The exosome suspension was prepared by suspending exosomes (equivalent to 5 μg of protein per mouse) derived from AD-MSCs cultured in Prime Surface Petri dishes in 25 μl of PBS. A hyaluronidase suspension was also prepared by suspending 100 U of hyaluronidase (Sigma-Aldrich) in 5 μl of PBS. 5 μl of the hyaluronidase suspension was administered intranasally to 6-month-old 5xFAD mice under anesthesia. Thirty minutes after administration of the hyaluronidase suspension, 5 μl of the exosome suspension or vehicle was administered intranasally five times every 5 minutes for a total of 25 μl. Similarly, the hyaluronidase suspension, exosome suspension, or vehicle was administered intranasally four times, every other week. Mice were tested for cognitive function one month after the initial administration (Figure 27).

[0162] Mice administered the exosome suspension or vehicle were subjected to a Y-maze test. The results are shown in Figure 28. As shown in Figure 28, the alternation rate (%) of mice in the group treated with exosomes derived from AD-MSCs cultured by Prime Surface was significantly higher than that of mice in the group treated with the vehicle (P<0.05, unpaired t-test).

[0163] Comparative Example 1: Administration experiment of exosomes derived from adipose-derived mesenchymal stem cells two-dimensionally cultured in a 2D dish Human AD-MSCs (Lonza) derived from a 33-year-old woman with a case of caucasoid were cultured on a two-dimensional culture support (150 mm cell culture dish, manufactured by BM Equipment Co., Ltd.) in a 20 ml cell suspension of AD-MSCs (1.0 x 10 6 The AD-MSCs were seeded with 2000kJ / ml (2000kcal) of 2000kJ / ml (1000kcal / ml) and cultured at 37°C for 3 days. A vehicle (PBS) and an exosome suspension were prepared. The AD-MSC culture medium was centrifuged to obtain a supernatant, which was then concentrated using an Amicon Ultra centrifugal ultrafiltration filter (Merck). Exosomes were isolated from the concentrated supernatant using the MagCapture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Corporation). The protein content of the isolated exosomes was measured using the BCA method (BCA Assay and Lowry Assays, Thermo Fisher Scientific). The exosome suspension was prepared by suspending exosomes derived from two-dimensionally cultured AD-MSCs (equivalent to 5 μg of protein per animal) in 25 μl of PBS. Additionally, a hyaluronidase suspension was prepared by suspending 100 U of hyaluronidase (Sigma-Aldrich) in 5 μl of PBS. Under anesthesia, 6-month-old 5xFAD mice were intranasally administered 5 μl of hyaluronidase suspension. Starting 30 minutes after administration of the hyaluronidase suspension, 5 μl of exosome suspension or vehicle was administered intranasally five times, every 5 minutes, for a total of 25 μl. Similarly, the hyaluronidase suspension, exosome suspension, or vehicle was intranasally administered four times, one week apart. One month after the initial administration, the mice were tested for cognitive function (Figure 29).

[0164] A Y-maze test was performed on mice administered with the exosome suspension or vehicle. The Y-maze test was performed in the same manner as in Example 3. The results are shown in Figure 30. As shown in Figure 30, there was no significant difference between the alternation behavior rate (%) of mice in the exosome group derived from AD-MSCs obtained by two-dimensional culture and the alternation behavior rate (%) of mice in the vehicle group (P = 0.0635, unpaired t-test). Example 12: Administration experiment of exosomes derived from transfected adipose-derived mesenchymal stem cells

[0165] Human AD-MSCs were transfected with 15 nm of a negative control miRNA mimic, miR-26a mimic, and let-7a mimic (all Thermo Fisher Scientific) using HiPerFect Transfection Reagent (QIAGEN). The human AD-MSCs were derived from a 33-year-old female with a case of caucasoid syndrome. Human AD-MSCs transfected with each microRNA mimic were cultured in 100 mm cell culture dishes (BM Instruments, Inc.). The culture supernatant was collected in the same manner as in Example 8, and exosomes were isolated from the culture supernatant using Magcapture™ Exosome Isolation kit PS (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0166] Exosome suspensions were prepared by suspending exosomes (1.6 μg protein / animal) derived from AD-MSCs transfected with each microRNA mimic in 25 μL of PBS. Hyaluronidase suspensions were also prepared by suspending 100 U of hyaluronidase (Sigma-Aldrich) in 5 μL of PBS. Under anesthesia, 13-15 month-old APP / PS1 mice were intranasally administered with 5 μL of hyaluronidase suspension. Starting 30 minutes after administration of the hyaluronidase suspension, 5 μL of each exosome suspension was administered intranasally five times, every 5 minutes, for a total of 25 μL. Similarly, hyaluronidase suspension and exosome suspension were intranasally administered four times, every other day. Four days after the initial administration, the mice were tested for cognitive function (Figure 31).

[0167] As shown in Figure 32, the alternation behavior rate (%) of mice fed with exosomes derived from AD-MSCs transfected with miR-26a mimic tended to be improved compared to the group of exosomes derived from AD-MSCs transfected with negative control miRNA mimic. The alternation behavior rate (%) of mice fed with exosomes derived from AD-MSCs transfected with let-7a mimic was significantly higher than that of the group of exosomes derived from AD-MSCs transfected with negative control miRNA mimic (P<0.05, unpaired t-test).

[0168] The present invention can be used to prevent or improve cognitive decline.

Claims

1. It contains a three-dimensional culture of mesenchymal stem cells as an active ingredient. A pharmaceutical composition for preventing or improving cognitive decline, characterized in that the three-dimensional culture of mesenchymal stem cells is a culture in which the expression level of at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells.

2. The pharmaceutical composition according to claim 1, characterized in that at least some of the uridine residues of the microRNA are replaced with pseudouridine residues.

3. The active ingredient is an exosome containing at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369. A pharmaceutical composition for preventing or improving cognitive decline, characterized in that the exosomes are derived from a three-dimensional culture of mesenchymal stem cells, and the content of at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to exosomes secreted from a two-dimensional culture of mesenchymal stem cells.

4. The pharmaceutical composition according to claim 1, characterized in that the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

5. The pharmaceutical composition according to claim 1, wherein the three-dimensional culture comprises a spheroid.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the cognitive decline is due to Alzheimer's disease.

7. A method for producing a three-dimensional culture of mesenchymal stem cells, The manufacturing method includes a culture step of culturing the mesenchymal stem cells in three dimensions using a culture carrier containing a hydrogel whose main component is a polysaccharide polymer, or a culture carrier whose main component is collagen, and The process includes a recovery step of separating the three-dimensional culture obtained in the culture step from the culture carrier and recovering the three-dimensional culture, A method for producing a three-dimensional culture of mesenchymal stem cells, characterized in that the three-dimensional culture is a culture in which the expression level of at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells.

8. A method for producing a three-dimensional culture of mesenchymal stem cells, The above manufacturing method includes a culture step of culturing spheroids as a three-dimensional culture using a spheroid culture plate, and The process includes a recovery step of recovering the three-dimensional culture obtained in the culture step from the spheroid culture plate, A method for producing a three-dimensional culture of mesenchymal stem cells, characterized in that the three-dimensional culture is a culture in which the expression level of at least one microRNA, let-7a, miR-26a, miR-27b, miR-34a, and miR-369, is increased compared to the two-dimensional culture of mesenchymal stem cells.

9. A method for producing a three-dimensional culture of mesenchymal stem cells according to claim 7 or 8, characterized in that the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

10. A method for producing exosomes, A step of producing a three-dimensional culture of mesenchymal stem cells by the manufacturing method described in claim 9, and A method for producing exosomes, characterized by including a separation step of separating exosomes from the three-dimensional culture.

11. A three-dimensional culture of mesenchymal stem cells, characterized by being produced by the manufacturing method described in claim 9.

12. An exosome characterized by being produced by the manufacturing method described in claim 10.

13. A step of producing a three-dimensional culture of mesenchymal stem cells by the manufacturing method described in claim 9, and A step of manufacturing a pharmaceutical composition for preventing or improving cognitive decline using the three-dimensional cultured material. A method for producing a pharmaceutical composition, characterized by containing [a certain substance].