CN118369416A

CN118369416A - Suspension culture method of adherent cells with stirring

Info

Publication number: CN118369416A
Application number: CN202280081987.7A
Authority: CN
Inventors: 岩上昌史; 畑中大辅; 木田克彦
Original assignee: Nissan Chemical Corp
Current assignee: Nissan Chemical Corp
Priority date: 2021-10-15
Filing date: 2022-10-14
Publication date: 2024-07-19

Abstract

The present invention provides a method for culturing adherent cells, comprising a step of culturing adherent cells in suspension in a medium comprising nanofibers composed of water-insoluble polysaccharides, wherein the culturing is performed with stirring.

Description

Suspension culture method of adherent cells with stirring

Technical Field

The present invention relates to a method for suspension culture of adherent cells with stirring, and the like.

Background

In recent years, methods for transplanting and injecting cells into living bodies have been developed mainly in the medical and cosmetic fields. Among them, adult stem cells and progenitor cells are attracting attention for reasons such as low risk of cancer formation and short differentiation time as compared with pluripotent stem cells.

When using these cells, a large number of cells in a good state are required, and as a method for providing these cells, a method of culturing stem cells or the like in a state of being adhered to microcarriers or the like and proliferating them is known.

However, the microcarriers currently generally available are pointed out as the following problems: since sedimentation occurs in the culture medium under static conditions, stirring is required during the culture, and cell death and the like are caused by collision of microcarriers with each other due to the stirring. In addition, the efficiency of cell proliferation is insufficient, and further improvement is desired.

The present inventors have developed a culture medium composition for culturing animal and plant cells and/or tissues in a suspended state by using nanofibers such as polysaccharides having improved dispersibility in water (patent document 1).

The inventors of the present application have also found that nanofibers composed of water-insoluble polysaccharides can be used as a carrier common to various operations such as i) suspension culture, ii) differentiation induction, iii) transportation or storage under non-freezing conditions, iv) transplantation, and v) recovery of physiologically active substances from culture supernatants (patent documents 2 and 3).

Prior art literature

Patent literature

Patent document 1: WO2015/111686

Patent document 2: WO2017/175751

Patent document 3: WO2018/182016

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a technique related to mass production of adherent cells such as adult stem cells and progenitor cells. Further, the present invention aims to provide a technique for efficiently producing adherent cells of better quality.

Means for solving the problems

The present inventors have made intensive studies to solve the above problems, and as a result, have found that it is possible to obtain adherent cells of good quality while promoting proliferation of the adherent cells by culturing adherent cells in suspension in a medium containing vitronectin-supported chitin nanofibers and chitosan nanofibers with stirring.

The inventors of the present application found that adherent cells cultured under these conditions formed spheres (sphere) of uniform size, and were highly undifferentiated and migratory.

The inventors of the present application have found that spheres formed under such conditions can be easily recovered by using a cell sieve, and that the spheres can be efficiently monocellular by using a cell dispersing agent.

Furthermore, the present inventors found that, in the mesenchymal stem cells cultured using the method of the present application, the expression of a specific gene was increased and the production ability of extracellular vesicles was promoted.

Furthermore, the present inventors studied a mechanism of promotion of the ability to produce extracellular vesicles in mesenchymal stem cells cultured using the method of the present application.

The inventors of the present application have found that proliferation of adherent cells can be promoted even under a condition in which a vitronectin-supported chitin nanofiber and stirring are combined (i.e., a condition in which a chitosan nanofiber is not used), and have further found a highly efficient passage method under such a condition.

Furthermore, the inventors of the present application confirmed that: in the agitation culture using no nanofibers and the agitation culture using only chitosan nanofibers, adherent cells do not proliferate sufficiently; the process of the application can also be carried out on a large scale.

Furthermore, the inventors of the present application confirmed the physical structure of nanofibers and adherent cells in spheres formed using the methods of the present application.

In addition, the inventors of the present application have found that the mesenchymal stem cells prepared using the method of the present application have a very high anti-inflammatory effect and, in addition, have a high therapeutic effect on arthropathy.

The inventors of the present application have further studied based on the above findings, and completed the present application.

Namely, the present invention is as follows.

[1] A method for culturing adherent cells, comprising a step of culturing adherent cells in suspension in a medium comprising nanofibers composed of water-insoluble polysaccharides, wherein the culture is performed with stirring.

[2] The method according to [1], wherein the stirring condition is a state in which nanofibers and cells are suspended in a medium, and a state in which the nanofibers and cells are continuously moved in a system by an external force.

[3] The method according to [1] or [2], wherein the stirring is performed by a device accompanied by a stirring blade, and the rotation speed thereof is 0.01 to 50.0 m/min in terms of blade tip speed.

[4] The method according to any one of [1] to [3], wherein the stirring is carried out continuously during the cell culture.

[5] The method according to any one of [1] to [4], wherein the amount of the nanofibers composed of the water-insoluble polysaccharide added to the medium is 0.0001 to 0.2% (w/v).

[6] The method according to any one of [1] to [5], wherein the nanofiber composed of the water-insoluble polysaccharide supports an extracellular matrix.

[7] The method according to any one of [1] to [6], wherein the water-insoluble polysaccharide is at least one selected from the group consisting of chitin, cellulose, and hemicellulose.

[8] The method of [6] or [7], wherein the extracellular matrix is at least one selected from the group consisting of collagen, fibronectin, vitronectin, laminin, RGD sequence, and cadherin.

[9] The method of any one of [1] to [8], wherein the adherent cells are selected from the group consisting of stem cells, progenitor cells, adult non-stem cells, primary cultured cells, cell lines, and cancer cells.

[10] The method of any one of [1] to [9], wherein the medium further comprises chitosan nanofibers.

[11] A method for producing spheres of adherent cells having a uniform sphere size, comprising a step of culturing adherent cells in suspension in a medium containing nanofibers composed of water-insoluble polysaccharides, wherein the culture is carried out with stirring.

[12] The method according to [11], wherein the stirring condition is a state in which nanofibers and cells are suspended in a medium, and a state in which the nanofibers and cells are continuously moved in the system by an external force.

[13] The method according to [11] or [12], wherein the stirring is performed by a device accompanied by a stirring blade, and the rotation speed thereof is 0.01 to 50.0 m/min in terms of blade tip speed.

[14] The method according to any one of [11] to [13], wherein the stirring is performed continuously during the cell culture.

[15] The method according to any one of [11] to [14], wherein the amount of the nanofibers composed of the water-insoluble polysaccharide added to the medium is 0.0001 to 0.2% (w/v).

[16] A method for separating spheres comprising the step of supplying a suspension of spheres produced by the method according to any one of [11] to [15] to a cell screen.

[17] A method of unicellular adherent cells in the form of spheres comprising:

step 1, performing suspension culture on adherent cells in a culture medium containing nanofibers composed of water-insoluble polysaccharides; the method comprises the steps of,

And 2, treating the spheres of the adherent cells obtained in the 1 with a cell dispersing agent.

[18] A mesenchymal stem cell in which the expression of at least one gene selected from the group consisting of CD55, HMOX1, TSPAN7, RAB27B, IL, GPX3, and MFAP4 is increased compared to a mesenchymal stem cell cultured by an adhesion culture.

[19] The mesenchymal stem cell of [18], wherein the production of extracellular vesicles is also promoted as compared to mesenchymal stem cells cultured by adherent culture.

[20] The mesenchymal stem cell of [19], wherein the extracellular vesicle is an exosome.

[21] A method for promoting the production of extracellular vesicles of mesenchymal stem cells, which comprises a step of culturing mesenchymal stem cells in suspension in a medium comprising nanofibers composed of a water-insoluble polysaccharide, wherein the culturing is performed with stirring.

[22] A method for producing mesenchymal stem cells in which production of extracellular vesicles is promoted, comprising a step of culturing mesenchymal stem cells in suspension in a medium comprising nanofibers composed of water-insoluble polysaccharides, wherein the culture is performed with stirring.

[23] The method of [21] or [22], wherein the extracellular vesicle is an exosome.

[24] A therapeutic agent for inflammatory diseases, which comprises the mesenchymal stem cell of any one of [18] to [20 ].

[25] A method for treating an inflammatory disease in a subject, comprising administering the mesenchymal stem cells of any one of [18] to [20] to a subject having an inflammatory disease.

[26] The use of the mesenchymal stem cell of any one of [18] to [20] in the manufacture of a medicament for treating an inflammatory disease.

[27] The mesenchymal stem cell of any one of [18] to [20], for use in the treatment of an inflammatory disease.

Effects of the invention

According to the present invention, adherent cells can be efficiently produced.

In addition, according to the present invention, spheres of adherent cells of uniform size can be produced. Furthermore, according to the present invention, adherent cells having an undifferentiated property and an enhanced migration property can be produced. Furthermore, according to the present invention, spheres having adherent cells of uniform size can be isolated. Furthermore, the spheres obtained by the present invention can be made into single cells extremely efficiently. In addition, according to the present invention, mesenchymal stem cells suitable for regenerative medicine, in which the ability to produce extracellular vesicles is promoted, can be produced.

Drawings

FIG. 1A photograph showing the observation of spheres of mesenchymal stem cells derived from human umbilical cord in suspension culture in a medium comprising vitronectin-supported chitin nanofibers and chitosan nanofibers under the respective conditions (conditions 4 and 5) of test example 3.

Fig. 2 is a diagram showing the result of image analysis (sphere extraction image) of the fluorescent dye image of fig. 1.

FIG. 3 is a graph showing the distribution of the sizes of spheres prepared under each condition (conditions 4 and 5) of test example 3.

FIG. 4 is a photograph showing the observation of spheres when mesenchymal stem cells derived from human umbilical cord were cultured in suspension in a medium comprising vitronectin-supported chitin nanofibers and chitosan nanofibers under each condition of test example 4.

Fig. 5 is a photograph of cells cultured in suspension under the conditions of test example 4 and then seeded in an orifice plate, using Cell3iMagerduos (manufactured by sceen holders co., ltd.).

Fig. 6 is a diagram showing a sphere extraction image obtained as a result of image analysis in test example 4.

FIG. 7 is a graph showing the number of spheres and the average diameter of spheres under each condition of test example 4.

Fig. 8 is a photograph showing the state of a sphere under each condition of test example 5.

FIG. 9 is a diagram showing the substrate and cells in the filtrate of test example 6.

FIG. 10A photograph showing the state of cells under each condition of test example 6.

FIG. 11A diagram showing a Rotea Single Use Kit connection pattern used for the dispersion of spheres and the collection of single cells in test example 7.

FIG. 12A photograph showing the state of spheres or single cells at each stage of test example 7.

FIG. 13A photograph showing the appearance of cells (day 0 and day 3 of culture) during the culture in test example 10.

FIG. 14 is a view showing microscopic observation images of cells (day 0 and day 3 of culture) during the culture in test example 10.

FIG. 15 is a diagram showing bright field images and fluorescent staining images of spheres or cells after each treatment in test example 11.

FIG. 16 is a view showing an image of a sphere or a cell used for analysis in test example 11.

FIG. 17A photograph of the spheres at each time point after the enzyme treatment was observed by using an inverted microscope in test example 12.

FIG. 18 is a photograph showing the state of each treated sphere or cell in test example 13 by using an inverted microscope.

FIG. 19 is a diagram showing that the enhanced expression of RAB27B protein in mesenchymal stem cells cultured by the method of the present invention was confirmed by Western blotting.

FIG. 20 is a diagram showing the confirmation of the expression of NFE2L2, P65, and phosphorylated P65 (P-P65) proteins in mesenchymal stem cells cultured by the method of the present invention by Western blotting.

FIG. 21 is a graph showing the confirmation of the expression level of RAB27B protein by Western blotting when various siRNA treatments were performed on mesenchymal stem cells cultured by the method of the present invention.

Fig. 22 is a graph showing the results of performing any one of the conditions 1 (fresh medium containing substrate 2 was simply added), 2 (fresh medium containing substrate 2 was added after the spheres were partially single-cellularized using physical shear force), and 3 (fresh medium containing no substrate was added) for passaging of the mesenchymal stem cells cultured using substrate 2.

FIG. 23 is a graph showing the results of passaging mesenchymal stem cells cultured using substrate 1 or substrate 2 by performing specific operations (operations 1 to 3).

FIG. 24 is a graph showing the results of suspension culture of mesenchymal stem cells using various substrates (substrate 1 to substrate 3) under stirring.

FIG. 25 is a diagram showing the shape of a sphere formed when the method of the present invention is carried out on a large scale (1L).

Fig. 26 is a view showing an image of a slice of a sphere prepared using the substrate 1 or the substrate 2.

Detailed Description

Hereinafter, the present invention will be described in detail.

1. Method for culturing adherent cells

The present invention provides a method for culturing adherent cells (hereinafter, sometimes referred to as "method of the present invention" or the like), comprising a step of culturing adherent cells in suspension in a medium containing nanofibers composed of water-insoluble polysaccharides, wherein the culturing is performed with stirring.

In the method of the present invention, adherent cells are cells that require a support such as a vessel wall during survival and proliferation.

In the method of the present invention, the adherent cells are not particularly limited, and examples thereof include stem cells, progenitor cells, adult non-stem cells, primary cultured cells, cell lines, and cancer cells. Stem cells are cells having both the ability to replicate itself and the ability to differentiate into cells of various other lineages. Examples of adherent stem cells include, but are not limited to, adult stem cells such as mesenchymal stem cells, neural stem cells, hematopoietic stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Mesenchymal stem cells refer to stem cells having the ability to differentiate into all or some of bone cells, cartilage cells and fat cells. Mesenchymal stem cells exist in tissues such as bone marrow, peripheral blood, umbilical cord blood, and adipose tissue at a low frequency, and can be isolated from these tissues by a known method. The progenitor cells are cells at a stage in the middle of differentiating from the stem cells into specific somatic cells and germ cells. Examples of adherent progenitor cells include, but are not limited to, precursor adipocytes, precursor cardiomyocytes, precursor endothelial cells, neural progenitor cells, hepatic progenitor cells, pancreatic progenitor cells, renal progenitor cells, and the like. Examples of adherent adult non-stem cells include, but are not limited to, fibroblasts, bone cells, periosteocytes, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglial cells, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, and the like. The primary cultured cells are cells which are in a state of being cultured after inoculating cells or tissues isolated from a living body until the first passage is performed. The primary cultured cells may be cells collected from any tissue such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain, or nerve tissue. The cell line is a cell that has been obtained by manual manipulation in vitro and has an unlimited proliferation capacity. The adherent cells in the methods of the invention are preferably stem cells or progenitor cells, more preferably mesenchymal stem cells.

The source of the adherent cells in the method of the present invention is not particularly limited, and may be any cells derived from animals and plants. The animal is not limited, and examples thereof include fish, amphibians, reptiles, birds, pancrustaceans, hexapods (Hexapoda), mammals, and the like, and preferably mammals. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, field mice, duckbill, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, macaque, chimpanzees, and humans. The plant is not particularly limited as long as the collected cells can be subjected to liquid culture. Examples thereof include plants (e.g., medicinal ginseng, vinca, scopolamine, coptis, belladonna, etc.) which produce crude drugs (e.g., saponins, alkaloids, berberine, scopolamine, phytosterol, etc.); plants (e.g., blueberries, safflower, madder, saffron, etc.) producing pigments and polysaccharides (e.g., anthocyanin, carthamin, alizarin, saffron, etc.) useful as raw materials for cosmetics and foods; or a plant producing a raw material of a drug, etc., but is not limited thereto.

In the present specification, nanofibers refer to fibers having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and still more preferably 0.01 to 0.02 μm.

In the method of the present invention, the aspect ratio (L/D) of the nanofibers to be used can be obtained from the average fiber length/average fiber diameter, and is not particularly limited, but is usually 2 to 500, preferably 5 to 300, more preferably 10 to 250.

In the present specification, the average fiber diameter (D) of the nanofibers is determined as follows. First, a collodion (Collodion) support film produced by Kagaku Co., ltd was hydrophilized for 3 minutes with Ion Cleaner (JIC-410) produced by Japanese electronics Co., ltd., and a plurality of drops of a nanofiber dispersion to be evaluated (diluted with ultrapure water) were added dropwise, followed by drying at room temperature. Transmission electron microscope (TEM, H-8000) (10,000 times) manufactured by Hitachi, inc. was used to observe the sample at an acceleration voltage of 200kV, and the obtained image was used for the number of samples: the fiber diameter of each nanofiber was measured for 200 to 250 nanofibers, and the arithmetic average thereof was taken as the average fiber diameter (D).

The average fiber length (L) was determined as follows. The nanofiber dispersion to be evaluated was diluted with pure water so as to be 100ppm, and nanofibers were uniformly dispersed using an ultrasonic cleaner. The nanofiber dispersion was cast (cast) onto a silicon wafer obtained by hydrophilizing the surface with concentrated sulfuric acid, and dried at 110℃for 1 hour to obtain a sample. Using an image of the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2,000 times) manufactured by japan electronics corporation, the number of samples was: 150 to 250 nanofibers were measured for each fiber length and the arithmetic average was taken as the average fiber length (L).

In a preferred mode, the nanofibers have the following effect: when mixed with a liquid medium, the nanofibers are uniformly dispersed in the liquid while maintaining the primary fiber diameter, and the viscosity of the liquid is not substantially increased, and the cells attached to the nanofibers are substantially maintained, thereby preventing sedimentation.

The nanofibers used in the method of the present invention are composed of water insoluble polysaccharides. The polysaccharide is a sugar polymer obtained by polymerizing 10 or more monosaccharides (for example, three-carbon sugar, four-carbon sugar, five-carbon sugar, six-carbon sugar, seven-carbon sugar, etc.).

Examples of the water-insoluble polysaccharide include celluloses such as cellulose and hemicellulose; chitin, chitosan, and the like, but are not limited thereto. The water-insoluble polysaccharide is preferably chitin or chitosan, more preferably chitin. In the present specification, the term "nanofiber composed of chitin" may be sometimes referred to as "chitin nanofiber". The same applies to other water-insoluble polysaccharides.

The chitin-based substance is 1 or more saccharides selected from the group consisting of chitin and chitosan. The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively, and generally, chitin substances which are high in N-acetylglucosamine content and are insoluble in acidic aqueous solutions are chitin, and chitin substances which are high in glucosamine content and are soluble in acidic aqueous solutions are chitosan. In the present specification, for convenience, the ratio of N-acetylglucosamine to the constituent sugar may be 50% or more, and less than 50% may be referred to as chitosan.

As the raw material of chitin, various biological resources such as shrimp, crab, insect, shellfish, mushroom, etc. can be used. The chitin used in the present invention may be chitin having an α -type crystal structure, such as chitin derived from crab shells and shrimp shells, or chitin having a β -type crystal structure, such as chitin derived from cuttlebone (cuttlebone). The shells of crabs and shrimps are often treated as industrial waste, and are preferable as raw materials from the viewpoint of easy availability and effective utilization, but a deproteinizing step and a deliming step are required for removing proteins, ash, and the like contained as impurities. Therefore, in the present invention, purified chitin which has been subjected to a matrix removal treatment is preferably used. Purified chitin is commercially available. The raw material of the chitin nanofiber used in the present invention may be chitin having any of alpha-type and beta-type crystal structures, and is preferably alpha-type chitin.

The polysaccharide nanofibers can be obtained by pulverizing the polysaccharide. The pulverizing method is not limited, and in order to achieve the fiber diameter and fiber length according to the object of the present invention, a method capable of obtaining a strong shearing force such as a high-pressure homogenizer, a grinding machine (stone mortar), or a medium stirring mill such as a bead mill is preferable.

Among them, the high-pressure homogenizer is preferably used for the pulverization, and it is desirable to use a wet pulverization method as disclosed in, for example, japanese patent application laid-open No. 2005-270891 and japanese patent No. 5232976 for the pulverization (pulverization). Specifically, the dispersion liquid obtained by dispersing the raw material is sprayed from a pair of nozzles at high pressure and is collided with each other, and the raw material is pulverized, for example, by Starburst System (high-pressure pulverizing device manufactured by Sugino MACHINE LIMITED) or NanoVater (high-pressure pulverizing device manufactured by Jifield mechanical Xingjingsu corporation).

When the raw material is pulverized (pulverized) using the aforementioned high-pressure homogenizer, the degree of pulverization and homogenization depends on the pressure to be fed to the ultrahigh-pressure chamber of the high-pressure homogenizer, the number of times the raw material passes through the ultrahigh-pressure chamber (the number of treatments), and the concentration of the raw material in the aqueous dispersion. The pressure-feed pressure (treatment pressure) is not particularly limited, but is usually 50 to 250MPa, preferably 100 to 200MPa.

The concentration of the raw material in the aqueous dispersion at the time of the micronization treatment is not particularly limited, but is usually 0.1 to 30% by mass, preferably 1 to 10% by mass. The number of treatments for the pulverization (pulverization) is not particularly limited, and if the concentration of the raw material is 0.1 to 1 mass%, the number of treatments is about 10 to 100 times, and if the concentration is 1 to 10 mass%, the number of treatments may be about 10 to 1000 times, depending on the concentration of the raw material in the aqueous dispersion.

The viscosity of the aqueous dispersion at the time of the above-mentioned fine treatment is not particularly limited, and in the case of, for example, α -chitin, the viscosity of the aqueous dispersion is in the range of 1 to 100mpa·s, preferably 1 to 85mpa·s (measured by tuning fork vibration viscosity at 25 ℃ (SV-1A,A&D Company Ltd.)). In the case of chitosan, the viscosity of the aqueous dispersion is in the range of 0.7 to 30 mPa.S, preferably 0.7 to 10 mPa.S (measured by tuning fork vibration viscosity at 25 ℃ C.) (SV-1A,A&D Company Ltd)).

The method for producing nanofibers is described in WO2015/111686A1 and the like.

In one embodiment, in the method of the present invention, the extracellular matrix may be supported on nanofibers composed of water-insoluble polysaccharides. In the present specification, the term "nanofiber-supported" as used herein refers to a state in which nanofibers are attached or adsorbed to an extracellular matrix without being chemically covalently bonded. The loading of the nanofiber-based extracellular matrix may be achieved by intermolecular forces, electrostatic interactions, hydrogen bonding, hydrophobic interactions, etc., but is not limited thereto. The state in which the nanofibers support the extracellular matrix may be referred to as a state in which the nanofibers are held in contact with the extracellular matrix without being chemically covalently bonded, or a state in which the nanofibers and the extracellular matrix form a complex without being chemically covalently bonded.

In the method of the present invention, the extracellular matrix supported on the nanofibers is not particularly limited as long as a desired effect can be obtained, and collagen (collagen I to XIX), fibronectin, vitronectin, laminin (laminin-1 to 12), RGD sequence, cadherin, and the like are exemplified. The choice of extracellular matrix varies depending on the kind of cells to be proliferated, and can be appropriately selected by those skilled in the art. Vitronectin is preferred as extracellular matrix, for example in the case of mesenchymal stem cells. In addition, in the case where vitronectin is human-derived vitronectin, vitronectin having the amino acid sequence of 20 to 398 (SEQ ID NO: 2) or 62 to 478 (SEQ ID NO: 1) is preferable. In the case of using vitronectin derived from a non-human, a region corresponding to a fragment of vitronectin derived from a human may be used.

In the method of the present invention, the amount of the extracellular matrix supported by the nanofibers is usually 0.001 to 50mg, preferably 0.01 to 10mg, more preferably 0.1 to 10mg, still more preferably 0.3 to 10mg, still more preferably 1 to 10mg, particularly preferably 2 to 10mg, per 1g of the extracellular matrix of the nanofibers, but the present invention is not limited thereto.

In the method of the present invention, the preparation of the nanofibers carrying the extracellular matrix may be performed as follows: the dispersion obtained by dispersing the nanofibers in an aqueous solvent is mixed with an aqueous solution of an extracellular matrix, and allowed to stand for a certain period of time as needed. Examples of the aqueous solvent for dispersing the nanofibers include, but are not limited to, water, dimethylsulfoxide (DMSO), and the like. As the aqueous solvent, water is preferable. Suitable buffers, salts may be included in the aqueous solvent. In order to uniformly contact the extracellular matrix with the nanofibers, it is preferable to perform sufficient mixing by a blowing operation or the like. Further, as the time of standing, the mixed solution of the dispersion liquid of the nanofibers and the aqueous solution of the extracellular matrix may be left standing for usually 30 minutes or longer, preferably 1 hour or longer, more preferably 3 hours or longer, still more preferably 6 hours or longer, still more preferably 9 hours or longer, and particularly preferably 12 hours or longer. The upper limit of the standing time is not particularly limited, and may be set to 48 hours or less (for example, 36 hours or less, 24 hours or less, 16 hours or less) as an upper limit. The temperature at the time of standing is not particularly limited, and may be usually 1 to 30 ℃, preferably 1 to 28 ℃,1 to 26 ℃,1 to 25 ℃,1 to 24 ℃,1 to 23 ℃,1 to 22 ℃,1 to 21 ℃,1 to 20 ℃,1 to 19 ℃,1 to 18 ℃,1 to 17 ℃,1 to 16 ℃ or 1 to 15 ℃, more preferably 2 to 10 ℃,5 to 25 ℃ or 15 to 25 ℃, particularly preferably 2 to 5 ℃ (e.g., 4 ℃) or 15 to 25 ℃ (e.g., 20 ℃).

The mixing ratio of the nanofibers composed of the water-insoluble polysaccharide to the extracellular matrix varies depending on the kind of the above-mentioned substances used, and can be set to, for example, 100:0.1 to 1, preferably 100:0.4 to 0.6, but is not limited thereto.

The amount of the extracellular matrix supported on the nanofibers composed of the water-insoluble polysaccharide can be measured by, for example, the Micro BCA method, the enzyme immunoassay (ELISA method), or the like, but is not limited thereto.

In a preferred embodiment, nanofibers composed of water-insoluble polysaccharides are uniformly dispersed in a liquid medium, and adherent cells attached to the nanofibers are suspended in the liquid medium.

In the method of the present invention, the medium containing the nanofibers supporting the extracellular matrix may be appropriately selected depending on the type of adherent cells used, and for example, when the culture of adherent cells of mammals is aimed, a medium generally used for the culture of mammalian cells may be used. Examples of the culture Medium for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), ham F12 Medium (Ham's NutrientMixture F) DMEM/F12 Medium, mcCoy5A Medium (McCoy's 5A Medium), eagle MEM Medium (Eagle's Minimum Essential Medium; EMEM), αmem medium (alphaModifiedEagles's Minimum Essential Medium; αmem), MEM Medium (MinimumEssential Medium), RPMI1640 Medium, modified duff Medium (Iscove's ModifiedDulbecco's Medium; IMDM), MCDB131 medium, william medium E, IPL medium, fischer's medium, stemPro (manufactured by Invitrogen), X-VIVO 10 (manufactured by Cambrex), X-VIVO 15 (manufactured by Cambrex), HPGM (manufactured by Cambrex), STEMSPAN H (manufactured by STEMCELL Technologies), STEMSPANSFEM (manufactured by STEMCELLTechnologies), and, STEMLINEII (SigmaAldrich Co., ltd.), QBSF-60 (Qualitybiological Co., ltd.), stemProhESCSFM (Invitrogen Co., ltd.), essential6 (registered trademark) medium (Gibco Co., ltd.) Essenal 8 (registered trademark) medium (manufactured by Gibco Co., ltd.), essenal 8 (registered trademark) Flex medium (manufactured by Thermo Fisher Co., ltd.), stemFlex medium (manufactured by Thermo Fisher Co., ltd.), STEMSCALE (registered trademark) PSC Suspension Medium (manufactured by Thermo Fisher Co., ltd.), mTESR1 or 2 or Plus medium (manufactured by STEMCELL Technologies Co., ltd.), REPRO FF or REPRO FF2 (manufactured by REPROCELL Co., ltd.), PSGro hESC/iPSC medium (manufactured by System Biosciences Co., ltd.), nutriStem (registered trademark) medium (manufactured by Biological Industries Co., ltd.), MSC NutriStem (registered trademark) XF Medium (Biological Industries Co., ltd.), CSTI-7 Medium (cell science research Co., ltd.), mesenPRO RS Medium (Gibco Co., ltd.), MF-Medium (registered trademark) mesenchymal stem cell proliferation Medium (Toyobo Co., ltd.), mesenchymal stem cell serum-free Medium (FUKOKU Co., ltd.), MESENCHYMAL STEM CELL Growth Medium2 (PromoCell Co., ltd.), Sf-900II (Invitrogen), opti-Pro (Invitrogen), stemFit (registered trademark) AK02N or Basic02 or AK03N or Basic03 or Basic04 medium (Ajinomoto Healthy Supply Co., ltd.), STEMUP medium (Nissan chemical Co., ltd.), and the like.

The above-mentioned medium may be freely added with sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugar, etc. by those skilled in the art according to the purpose. In culturing mammalian cells, one skilled in the art may add one or more other chemical components or biological components in combination according to the purpose. Examples of components that can be added to the medium for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, thioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules. Examples of cytokines that can be added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon alpha (IFN-alpha), interferon beta (IFN-beta), interferon gamma (GM-gamma), cytokine G (FL), granulocyte colony-stimulating factor (SCF), granulocyte colony-stimulating factor (CSF), and granulocyte colony-stimulating factor (SCF-colony-stimulating factor) and granulocyte colony-stimulating factor (Fl-2) Leukemia cell inhibitory factor (LIF), oncomelanin M (OM), erythropoietin (EPO), thrombopoietin (TPO), etc., but are not limited thereto.

As a hormone which can be added to the medium, examples include melatonin, 5-hydroxytryptamine, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mu Leshi-tube hormone, adiponectin, corticotropin, angiotensinogen, angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental prolactin, growth hormone, inhibin, insulin-like growth factor leptin, luteinizing hormone, melanocyte stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin (thiombopoetin), thyroid stimulating hormone releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin releasing hormone, lipotropin (lipotropin), brain natriuretic peptide, neuropeptides Y, histamine, endothelin, pancreatic polypeptide, renin and enkephalin, but are not limited to these.

Examples of growth factors that can be added to the medium include transforming growth factor- α (TGF- α), transforming growth factor- β (TGF- β), macrophage inflammatory protein-1α (MIP-1α), epithelial cell growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve cell growth factor (NGF), hepatocyte Growth Factor (HGF), leukemia Inhibitory Factor (LIF), protease connexin I, protease connexin II, platelet-derived growth factor (PDGF), cholinergic Differentiation Factor (CDF), chemokines, notch ligand (Delta 1, etc.), wnt proteins, angiopoietin-like proteins 2, 3, 5, or 7 (Angpt, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), pleiotrophin (Pleiotrophin), and the like.

In addition, a substance obtained by artificially changing the amino acid sequences of these cytokines and growth factors by gene recombination techniques may be added. Examples thereof include IL-6/soluble IL-6 receptor complex and Hyper IL-6 (fusion protein of IL-6 and soluble IL-6 receptor).

Examples of antibiotics that can be added to the medium include sulfanilamide preparations, penicillin, fenesillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclopillin, carbenicillin, ticarcillin, piperacillin, azlocillin, meloxicam, amoxicillin, adexillin (andinocillin), cephalosporins and derivatives thereof, oxaquin, ofloxacin, temafloxacin, nalidixic acid, pyrrolimic acid, ciprofloxacin, cinnoxacin, norfloxacin, mefloxacin, rosaxacin, ofloxacin, enoxacin, piprazole, sulbactam, clavulanic acid, beta-bromopenicillin acid (beta-bromopenicillanic acid), beta-clopenicillic acid (beta-chloropenicillanic acid), 6-acetylmethylene-penicillic acid, cefprozin, sulbazole, adinoshirin and amoxazole (andinocillin), and derivatives thereof, and the other forms of the drugs, such as, and the drugs, may be completely taken from the drugs.

In one embodiment, supplements, serum substitutes may be added to the medium. Examples of these include StemPro (registered trademark) Neural Supplement (manufactured by Thermo Fisher corporation), B-27 (registered trademark) support (manufactured by Thermo Fisher corporation), knockOut (registered trademark) Serum Replacement (manufactured by Thermo Fisher corporation), CTS (registered trademark) KnockOut (registered trademark) SR XenoFree Medium (manufactured by Thermo Fisher corporation), ELAREM (registered trademark) PRIME I RESEARCH GRADE (manufactured by PL Bioscience corporation), ELAREM (registered trademark) ELAREM or GMP Grade (manufactured by PL Bioscience corporation), ELAREM (registered trademark) ELAREM-ELAREM or GMP Grade (manufactured by PL Bioscience corporation), ELAREM-ELAREM (manufactured by PL Bioscience corporation), human ELAREM (manufactured by ELAREM), ELAREM-Reduced Human (manufactured by ELAREM corporation), T-2 (registered trademark) (manufactured by ELAREM corporation) 2 (manufactured by ELAREM-Reduced Human) or (registered trademark) (ELAREM) (manufactured by the company), and ELAREM (manufactured by ELAREM-ELAREM or ELAREM-ELAREM (manufactured by the company) (ELAREM-ELAREM or ELAREM) (the ELAREM-3-registered trademark), ELAREM (manufactured by the ELAREM corporation) and ELAREM (manufactured by the ELAREM-ELAREM or ELAREM).

Examples of cell adhesion molecules that can be added to the medium include, but are not limited to, a Vitronectin (VTN-N) recombinant human protein, a truncated version (manufactured by Thermo Fisher Co., ltd.), a CTS (registered trademark) Vitronectin (VTN-N) recombinant human protein, a truncated version (manufactured by Thermo Fisher Co., ltd.), RHLAMININ-521 (manufactured by Thermo Fisher Co., ltd.), iMatrix-511MG (manufactured by Matrixome Co., ltd.), iMatrix-511silk or-411 or-221 (manufactured by Matrixome Co., ltd.), nutriCoat (manufactured by registered trademark) ATTACHMENT SOLUTION (manufactured by Biological Industries Co., ltd.).

The mixing ratio is not particularly limited, and the dispersion of nanofibers: liquid medium (aqueous medium) (volume ratio) is typically 1:99 to 99:1, preferably 10: 90-90: 10, more preferably 20: 80-80: 20.

In the present specification, the term "suspension of cells" means a state in which cells are not adhered to a culture vessel (non-adhesion), regardless of whether or not cells are settled.

The adherent cells can be cultured in suspension by culturing the adherent cells under agitation in a state of being attached to nanofibers capable of supporting an extracellular matrix. Since the substrate composed of nanofibers capable of supporting an extracellular matrix is dispersed so as not to be dissolved in a liquid medium or to be adhered to a culture vessel, when adherent cells are cultured in the liquid medium with stirring, the adherent cells adhere to the substrate and are uniformly suspended in the medium.

When the adherent cells are cultured in suspension using a substrate composed of nanofibers capable of supporting an extracellular matrix, the adherent cells prepared separately are added to a medium composition containing the substrate and uniformly mixed. The mixing method in this case is not particularly limited, and examples thereof include mixing by hand such as blowing (pipetting), and mixing using a device such as a stirrer, a vortex mixer, a microplate mixer, or a shaker.

After mixing the culture medium with adherent cells, the resulting cell suspension was cultured while stirring.

In the present invention, the term "agitation" refers to a state in which a substrate such as fiber and cells are suspended in a medium, and a state in which the substrate and cells continuously move in a system by an external force. The substrate and the cells are appropriately brought into contact with each other by an external force in the medium, thereby promoting the formation of a cell mass in which the substrate is incorporated, and allowing the cells to proliferate efficiently. The external force applied to the system may be appropriately adjusted according to the substrate concentration, the culture scale, etc., and a smooth mixing to such an extent that the cells are not damaged is preferable. The method of applying the external force includes

(1) Mixing based on rotation of stirring blade,

(2) Oscillating and mixing in a reciprocating type, a rotary type (for example, a mode in which the culture vessel rotates in the horizontal direction), a teeter-totter type, a wave-like shaking type (WAVE SHAKING TYPE), etc,

(3) Mixing based on reflux or gas aeration;

(4) Rotary mixing using roller bottles; or (b)

(5) Mixing by vibration based vortex mixer; and the like,

The external force application method is not particularly limited as long as the uniform contact between the substrate and the cells is promoted by applying a moderate external force, and as a result, the formation of the cell mass including the substrate is promoted.

The term "uniform" as used herein refers to a state in which the substrate and cells are suspended at a macroscopic angle in which they are not biased to exist on the bottom surface of the vessel and remain stationary, and does not mean that the substrate and cells are uniformly distributed throughout the culture medium at a microscopic angle.

The agitation of the liquid medium may be carried out by a method known per se. For example, a magnetic stirrer, stirring blade, or the like may be exemplified, but is not limited thereto. The shape of the stirring blades used for stirring, the number of stirring blades, the rotational speed and the frequency thereof may be appropriately set according to the purpose of those skilled in the art. The lower limit of the stirring speed (that is, blade tip speed) used in the present invention is not particularly limited as long as the cells and the substrate are not stationary, and is, for example, usually 0.01 m/min or more, preferably 0.10 m/min or more (for example, 0.15 m/min or more), and more preferably 0.90 m/min or more (for example, 0.97 m/min). The upper limit thereof may be, for example, generally 50.0 m/min or less, preferably 30.0 m/min or less (e.g., 22.6 m/min), more preferably 20.00 m/min or less (e.g., 15.08 m/min). In one aspect of the present invention, the blade tip speed may be generally 0.01 to 50.0 m/min, preferably 0.10 to 30.0 m/min (e.g., 0.15 to 22.6 m/min), and more preferably 0.90 to 16.00 m/min.

In one embodiment, the lower limit of the stirring speed (i.e., the rotation speed) used in the present invention is not particularly limited as long as the cells and the substrate are in a non-stationary state, and is, for example, usually 1rpm or more, preferably 5rpm or more, and more preferably 10rpm or more. The upper limit thereof may be, for example, generally 150rpm or less, preferably 140rpm or less, and more preferably 120rpm or less. In one embodiment of the present invention, the stirring speed may be generally 1 to 150rpm, preferably 5 to 140rpm, and more preferably 10 to 120rpm.

The frequency of stirring may be any frequency as long as the desired effect of the present invention is obtained. For example, the stirring at a specific rotation speed selected from the above rotation speeds may be repeated for 1 cycle with no stirring for 1 minute and 59 minutes during the cell culture. Or may be stirred continuously during cell culture.

The temperature at which the cells are cultured is usually 25 to 39℃in the case of animal cells, preferably 33 to 39℃at (for example, 37 ℃). For the CO ₂ concentration, it is usually 4 to 10% by volume, preferably 4 to 6% by volume, in the atmosphere of the culture. The concentration of dissolved oxygen in the medium may be appropriately set according to the type of cells and the purpose of the culture. The pH in culturing the cells may be appropriately set depending on the cell type and the purpose of the culture, and in the case of animal cells, the pH is usually 7 to 8, preferably 7.2 to 7.8. In order to maintain the pH, the amount and concentration of CO ₂ added to the culture system may be adjusted, or an acid or alkali solution may be added. In addition, a nutrient source (for example, glucose) for the cells may be added appropriately, or the cells may be cultured while only waste (for example, lactic acid) is removed by using a membrane or the like. The culture time may be appropriately set according to the purpose of the culture.

The adherent cells in the method of the present invention can be cultured using culture vessels such as dishes, flasks, plastic bags, teflon (registered trademark) bags, dishes, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-well plates, chamber slides, tubes, trays, culture bags, roller bottles, etc., which are commonly used for culturing cells. These culture vessels are expected to be cell-low adherent so that adherent cells adhering to the substrate used in the present invention do not adhere to the culture vessel. As the culture vessel with low adhesion to cells, a culture vessel whose surface is not artificially treated for the purpose of improving adhesion to cells (for example, coating treatment with extracellular matrix or the like), or a culture vessel whose surface is artificially treated for the purpose of reducing adhesion to cells can be used.

When the medium is to be replaced, the stirring is stopped to naturally settle the cells and the substrate, and only the supernatant may be replaced. Or separating cells by centrifugation and filtration, and adding fresh culture medium to the cells. Alternatively, after the cells are properly concentrated by centrifugation and filtration, a fresh medium may be added to the concentrated solution. For example, the gravitational acceleration (G) at the time of centrifugation is 100 to 400G, and the size of the fine pores of the filter used at the time of the filtration treatment is 10 μm to 100 μm, but is not limited thereto.

The adherent cells can be cultured by a bioreactor or an automatic culture apparatus capable of automatically inoculating cells, changing a medium, obtaining an image of the cells, recovering the cultured cells, controlling pH, temperature, oxygen concentration, etc. under mechanical control and in a closed environment, while achieving high-density culture.

When suspension culture is performed with stirring in a state in which adherent cells are attached to a substrate composed of nanofibers capable of supporting an extracellular matrix, the adherent cells efficiently proliferate in the form of spheres. In the case where the adherent cells are stem cells such as mesenchymal stem cells, the cells obtained by this method have increased expression of genes such as undifferentiated markers (OCT 4, NANOG, etc.) and homing/migration markers (CXCR 4, etc.). That is, the adherent cells (e.g., mesenchymal stem cells) obtained in the present invention can be suitably used as cells for living body transplantation, for example. In addition, the spheres obtained in the present invention tend to have a uniform size distribution.

When the adherent cells are grown in suspension with the adherent cells attached to a substrate made of nanofibers capable of supporting an extracellular matrix, a medium capable of growing the adherent cells while maintaining the properties of the cells is used as a medium used for the suspension. The medium may be appropriately selected by those skilled in the art according to the kind of adherent cells.

In one embodiment, the culture medium used in the method of the present invention may contain chitosan nanofibers in addition to nanofibers that can support an extracellular matrix.

The chitosan nanofiber used in the method of the present invention may use the chitosan nanofiber prepared according to the above-mentioned nanofiber preparation method. Alternatively, commercially available chitosan nanofibers may be used.

In one embodiment of the present invention, when only nanofibers composed of water-insoluble polysaccharides are added to a liquid medium, the amount of nanofibers composed of water-insoluble polysaccharides (e.g., chitin nanofibers) added to the medium is not particularly limited as long as the desired effect can be obtained, and the nanofibers may be blended in the liquid medium in a range of usually 0.0001 to 0.2% (w/v), preferably 0.0005 to 0.1% (w/v), more preferably 0.001 to 0.07% (w/v), and particularly preferably 0.003 to 0.05% (w/v).

In one embodiment of the method of the present invention, when nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers that do not support an extracellular matrix are added to a liquid medium, nanofibers composed of water-insoluble polysaccharide are used in order to prepare a medium containing nanofibers composed of water-insoluble polysaccharide (e.g., chitin nanofibers) and chitosan nanofibers at a desired ratio (by weight): chitosan nanofiber = 1:0.01 to 10 (preferably nanofibers composed of water-insoluble polysaccharide: chitosan nanofibers=1:0.02 to 9, more preferably nanofibers composed of water-insoluble polysaccharide: chitosan nanofibers=1:0.05 to 8, still more preferably nanofibers composed of water-insoluble polysaccharide: chitosan nanofibers=1:0.1 to 7, still more preferably nanofibers composed of water-insoluble polysaccharide: chitosan nanofibers=1:0.5 to 6, particularly preferably nanofibers composed of water-insoluble polysaccharide: chitosan nanofibers=1:1 to 5). The resulting mixture of nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers may be mixed in a liquid medium so that the concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers) contained in the medium is usually 0.0001 to 0.2% (w/v), preferably 0.0005 to 0.1% (w/v), more preferably 0.001 to 0.07% (w/v), and particularly preferably 0.003 to 0.05% (w/v). Alternatively, a desired culture medium may be prepared by adding a desired amount of nanofibers composed of water-insoluble polysaccharide (e.g., chitin nanofibers) and chitosan nanofibers to a liquid culture medium, respectively, and stirring them well.

In one embodiment, the concentration of the nanofibers (e.g., chitin nanofibers) and chitosan nanofibers in the method of the present invention, which are composed of water insoluble polysaccharides, satisfies the following conditions: (1) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.0001 to 0.2% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(2) The concentration of the total nanofibers (nanofibers composed of water insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.0005 to 0.1% (w/v), and the nanofibers composed of water insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(3) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the medium composition is 0.001 to 0.05% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

Or alternatively

(4) The concentration of the total nanofibers (nanofibers composed of water insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.003 to 0.05% (w/v), and the nanofibers composed of water insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6).

In another embodiment, the obtained mixture of nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers may be mixed in a liquid medium so that the concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers) contained in the medium is usually 0.0001 to 1.0% (w/v), preferably 0.001 to 0.5% (w/v), more preferably 0.002 to 0.3% (w/v), and particularly preferably 0.003 to 0.1% (w/v).

In another embodiment, the concentration of the nanofibers (e.g., chitin nanofibers) and chitosan nanofibers in the method of the present invention, which are composed of water insoluble polysaccharides, satisfies the following conditions:

(5) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.0001 to 1.0% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(6) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the medium composition is 0.001 to 0.5% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(7) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.005 to 0.3% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

Or alternatively

(8) The concentration of the total nanofibers (nanofibers composed of water-insoluble polysaccharides and chitosan nanofibers) contained in the culture medium composition is 0.01 to 0.1% (w/v), and the nanofibers composed of water-insoluble polysaccharides contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6).

In the method of the present invention, when nanofibers composed of water-insoluble polysaccharide and chitosan nanofibers supporting an extracellular matrix are added to a liquid medium, the nanofibers supporting an extracellular matrix (e.g., vitronectin) may be used as the nanofibers supporting an extracellular matrix in order to prepare a medium containing the nanofibers supporting an extracellular matrix (e.g., chitin nanofibers) and chitosan nanofibers at a desired ratio (by weight): chitosan nanofiber = 1:0.01 to 10 (preferably, nanofibers supporting extracellular matrix: chitosan nanofibers=1:0.02 to 9, more preferably, nanofibers supporting extracellular matrix: chitosan nanofibers=1:0.05 to 8, still more preferably, nanofibers supporting extracellular matrix: chitosan nanofibers=1:0.1 to 7, still more preferably, nanofibers supporting extracellular matrix: chitosan nanofibers=1:0.5 to 6, particularly preferably, nanofibers supporting extracellular matrix: chitosan nanofibers=1:1 to 5). The resulting mixture of nanofibers/chitosan nanofibers supporting extracellular matrix may be mixed in a liquid medium so that the concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the medium is usually 0.0001 to 0.2% (w/v), preferably 0.0005 to 0.1% (w/v), more preferably 0.001 to 0.07% (w/v), and particularly preferably 0.003 to 0.05% (w/v). Alternatively, a desired culture medium may be prepared by adding a desired amount of nanofibers (e.g., chitin nanofibers) and chitosan nanofibers supporting an extracellular matrix (e.g., vitronectin) to a liquid culture medium, respectively, and stirring them well.

In one embodiment, the concentration of nanofibers (e.g., chitin nanofibers) and chitosan nanofibers carrying an extracellular matrix (e.g., vitronectin) in the method of the present invention satisfies the following conditions:

(1) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the culture medium composition is 0.0001 to 0.2% (w/v), and the nanofibers supporting extracellular matrix contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(2) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the medium composition is 0.0005 to 0.1% (w/v), and the nanofibers supporting extracellular matrix contained in the medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(3) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the medium composition is 0.001 to 0.05% (w/v), and the nanofibers supporting extracellular matrix contained in the medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

Or alternatively

(4) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the culture medium composition is 0.003 to 0.05% (w/v), and the nanofibers supporting extracellular matrix contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6).

In another embodiment, the resultant mixture of nanofibers/chitosan nanofibers supporting extracellular matrix may be mixed in a liquid medium so that the concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the medium is usually 0.0001 to 1.0% (w/v), preferably 0.001 to 0.5% (w/v), more preferably 0.002 to 0.3% (w/v), and particularly preferably 0.003 to 0.1% (w/v).

In another aspect, the concentration of nanofibers (e.g., chitin nanofibers) and chitosan nanofibers carrying an extracellular matrix (e.g., vitronectin) in the methods of the present invention satisfies the following conditions:

(5) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the culture medium composition is 0.0001 to 1.0% (w/v), and the nanofibers supporting extracellular matrix contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(6) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the medium composition is 0.001 to 0.5% (w/v), and the nanofibers supporting extracellular matrix contained in the medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

(7) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the culture medium composition is 0.005 to 0.3% (w/v), and the nanofibers supporting extracellular matrix contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6);

Or alternatively

(8) The concentration of the total nanofibers (nanofibers supporting extracellular matrix and chitosan nanofibers) contained in the culture medium composition is 0.01 to 0.1% (w/v), and the nanofibers supporting extracellular matrix contained in the culture medium composition: the weight ratio of the chitosan nanofiber is 1:0.01 to 10 (preferably 1:0.02 to 9, 1:0.05 to 8, 1:0.1 to 7, 1:0.5 to 7, or 1:1 to 6).

In one embodiment, polysaccharides having an effect of suspending cells and tissues may be used in combination. Examples of the polysaccharide include, but are not limited to, hyaluronic acid, gellan gum, deacylated gellan gum, neutral gum, diutan gum, xanthan gum (xanthogen gum), carrageenan, xanthan gum (zanthan gum), hexuronic acid, fucoidan, pectin, pectic acid, heparan sulfate, heparin, heparan sulfate, keratan sulfate, chondroitin sulfate, dermatan sulfate, rhamnosan sulfate, and salts thereof. One kind of these polysaccharides may be used, or two or more kinds may be used.

2. Method for producing spheres of adherent cells having uniform sphere size

The present invention also provides a method for producing spheres of adherent cells having a uniform sphere size (hereinafter, sometimes referred to as "the production method of the present invention"), comprising a step of culturing adherent cells in suspension in a medium containing nanofibers composed of water-insoluble polysaccharides, wherein the culturing is performed with stirring. The uniformity of the sphere size is important from the viewpoint of, for example, making the quality of the spheroid preparation uniform.

The method for producing the present invention is characterized by comprising nanofibers composed of water-insoluble polysaccharides. The manufacturing method of the present invention focuses on the uniformity of spheres produced by the method of the present invention. Therefore, the manufacturing method of the present invention is identical in constitution to the method of the present invention. Accordingly, the respective corresponding configurations of the production method of the present invention can refer to the configurations described in the method of the present invention. For example, nanofibers composed of water-insoluble polysaccharides, chitosan nanofibers, and extracellular matrices in the production method of the present invention are the same as those described in the method of the present invention.

3. Separation method of spheres

The present invention also provides a method for separating spheres (hereinafter, sometimes referred to as "the separation method of the present invention"), which comprises a step of supplying a suspension of spheres produced by the production method of the present invention to a cell screen.

The pore size of the mesh (mesh) of the cell sieve used in the separation method of the present invention is not particularly limited as long as it is smaller than the size of the sphere to be recovered, and may be generally 20 to 600. Mu.m, preferably 20 to 550. Mu.m, 20 to 500. Mu.m, 20 to 450. Mu.m, 20 to 400. Mu.m, 20 to 350. Mu.m, more preferably 30 to 350. Mu.m, 30 to 300. Mu.m, 30 to 280. Mu.m, 30 to 250. Mu.m, 30 to 230. Mu.m, particularly preferably 50 to 250. Mu.m, 60 to 230. Mu.m, 60 to 220. Mu.m.

The cell sieve used in the separation method of the present invention may be commercially available. As an example, a cell screen manufactured by pluriSelect corporation used in the following examples can be preferably used, and is not particularly limited. For the scale-up, HARVESTAINER (manufactured by Thermo FISHER SCIENTIFIC), which is a large-sized bag-type cell screen, a cell screen having a similar function to the above, CTS Rotea Counterflow Centrifugation System (manufactured by Thermo FISHER SCIENTIFIC) and Ksep (manufactured by Sartorius) Systems (manufactured by Sartorius) which are continuous elutriation Systems capable of separating spheres of a desired size in terms of size and specific gravity are used.

The conditions for passing the suspension of spheres through the cell screen are not particularly limited, and may be any method known per se or an instruction provided by the manufacturer of the cell screen.

The spheres produced by the production method of the present invention are a mixture with nanofibers or the like composed of water-insoluble polysaccharides. By using the separation method of the present invention, spheres can be efficiently separated from the mixture.

4. Method for single-cell formation of sphere

The invention also provides a method for unicellular adherent cells in the form of spheres. (hereinafter, sometimes referred to as "the method for single-cell formation of the present invention"), which comprises:

The nanofibers composed of water-insoluble polysaccharides used in step 1 of the single-cell method of the present invention are the same as those described in the method of the present invention.

In step 1 of the single-cell method of the present invention, the suspension culture of adherent cells may be performed under static conditions or under stirring conditions. When the stirring is carried out under stirring conditions, various parameters may be appropriately employed as described in the method of the present invention.

In one embodiment, in step 1 of the single-cell method of the present invention, the extracellular matrix may be supported on a nanofiber composed of a water-insoluble polysaccharide. The extracellular matrix, its loading, etc. are the same as those described in the method of the present invention.

In one embodiment, in step1 of the single-cell method of the present invention, chitosan nanofibers may be further added to the medium. The amount of chitosan nanofibers used and the like are the same as those described in the method of the present invention.

The other conditions (type of adherent cells, culture vessel, additional components, etc.) related to the suspension culture of adherent cells in the single-cell method of the present invention are the same as those described in the method of the present invention.

The cell dispersing agent that can be used in the method for single-cell formation of the present invention is not particularly limited as long as it can disperse spheres of adherent cells. Examples of the cell dispersing agent include enzymes having a cell dispersing action such as trypsin, collagenase, dispase, thermolysin, papain, hyaluronidase, elastase, pronase, and enzymes that decompose extracellular matrix. As the cell dispersing agent, a chelating agent such as EDTA can be used. In addition, as for the cell dispersion agent, a plurality of enzymes may be used in the form of a mixture (cocktail), or the enzymes may be used in combination with a chelating agent. The enzyme that breaks down nanofibers may be used in combination with chitinase, lysozyme or the like, or fine particles such as silica as an additive that promotes the breaking down reaction. The amount and concentration of the cell dispersant and the chelating agent to be added may be appropriately adjusted, and if the spheres are large and difficult to disperse, the amount of the enzyme to be added may be increased or the concentration may be increased. The cell dispersion agent can be prepared by a method known per se, and a commercially available cell dispersion agent can be used. Examples of the commercially available cell dispersion agent include Liberase (registered trademark) TM, TL, DL, DH, TH (manufactured by Merck corporation), liberase MNP-S, liberase MTFC/T, liberase T-Flex (manufactured by Roche Diagnostics corporation), TRYPLE SELECT Enzyme (manufactured by Thermo FISHER SCIENTIFIC corporation), hyQTase enzymatic CELL DETACHMENT solution (manufactured by Cytiva corporation), and, Accutase (registered trademark), accumax (registered trademark), accutaseLZ (registered trademark) (manufactured by InnovativeCell Technologies company), reLeSR (registered trademark), GENTLE CELL Dissociation Reagent (manufactured by STEMCELL Technologies company), zymeFree (registered trademark) Enzyme Free Cell Dissociation Reagent (manufactured by HiMedia Laboratories company), Collagenase, collagenase/Elastase, collagenase, types 1 to 7, STEMxyme (registered trademark) 1, STEMxyme (registered trademark) 2, collagenase, types A to C, neutral Protease (dispese), elastase (manufactured by Worthington biochemical corporation corporation), dispese (manufactured by Thermo FISHER SCIENTIFIC corporation), and, BD horizons (registered trademark) Dri Tumor & Tissue Dissociation Reagent (TTDR) (manufactured by BD Co., ltd.), chitinase 18a (manufactured by nzytech Co., ltd.), chitinase 18a from Bacillus licheniformis,Recombinant (manufactured by Creative Enzymes Co., ltd.), CHITINASE (CLOSTRIDIUM THERMOCELLUM) (manufactured by Megazyme Co., ltd.), Lysozyme, egg white (manufactured by Fuji photo-pure chemical Co., ltd.), japanese pharmacopoeia lysozyme standard (manufactured by Fuji photo-pure chemical Co., ltd.), etc., but are not limited thereto.

The various conditions (treatment temperature, treatment time, etc.) for treating the spheres of adherent cells with the cell dispersion agent can be appropriately set by those skilled in the art according to the kind of cell dispersion agent used. The treatment time may be usually 5 seconds to 60 minutes, preferably 10 seconds to 50 minutes, 30 seconds to 40 minutes, and more preferably 1 minute to 30 minutes. The treatment temperature may be usually set to 0 to 70 ℃, preferably 5 to 50 ℃, and more preferably 10 to 40 ℃.

In order to suppress adverse effects on cells (for example, cell death, viscosity increase, etc.) caused by cell dispersion, a ROCK inhibitor such as Y-27632, DNaseI, etc. may be added together with the cell dispersion agent at the time of single-cell formation or appropriately after single-cell formation.

In one embodiment, the method for single-cell formation of the present invention may be a method for single-cell formation of adherent cells in the form of spheres, which comprises a step of treating spheres of adherent cells obtained by the above-described method of the present invention, the method for producing the present invention, or the method for separating the present invention with a cell dispersion agent.

The single-cell process is not particularly limited as long as it is a single cell in a state where the cells live, and may be performed in a culture bag or a bioreactor in a state of standing or stirring. In addition, "cell dispersion tool" (manufactured by ABLE Corporation) as a means for single-cell formation of cells may be used for single-cell formation. The single cells may be formed by circulating in a channel of CTS Rotea Counterflow Centrifugation System (manufactured by Thermo FISHER SCIENTIFIC) or Ksep (registered trademark) Systems (manufactured by Sartorius).

While not wishing to be bound by theory, it is thought that the spheres of adherent cells prepared by a suspension culture method without using a substrate such as nanofibers are cell masses containing only adherent cells, and that the cells adhere strongly to each other in the cell masses, and therefore, a dispersion treatment with high strength is required to single-cell the cell masses. However, the dispersion treatment with high intensity damages cells, and thus the number of living single-cell-treated cells obtained is reduced. On the other hand, the cell mass composed of the adherent cells and the base material such as nanofibers can be subjected to a relatively smooth dispersion treatment. As a result, it is considered that the single-celled adherent cells can be efficiently produced.

5. Mesenchymal stem cells with enhanced expression of specific genes

The present invention also provides a mesenchymal stem cell in which expression of a specific gene is enhanced as compared with a mesenchymal stem cell cultured by an adherent culture (hereinafter, sometimes referred to as "mesenchymal stem cell of the present invention"). The mesenchymal stem cells of the present invention may be prepared by culturing the mesenchymal stem cells using the method of the present invention described above. The mesenchymal stem cells of the present invention are cultured in a suspended state using nanofibers or the like composed of water-insoluble polysaccharides under stirring conditions, thereby becoming mesenchymal stem cells having a gene expression profile different from that of the mesenchymal stem cells subjected to the adhesive culture.

In the mesenchymal stem cells of the present invention, CD55(NCBI Gene ID：1604)、HMOX1(NCBI Gene ID：3162)、TSPAN7(NCBI Gene ID：7102)、RAB27B(NCBI Gene ID：5874)、IL33(NCBI Gene ID：90865)、GPX3(NCBI Gene ID：2878)、 or MFAP4 (NCBI Gene ID: 4239) is exemplified as the Gene whose expression is enhanced.

The gene whose expression is enhanced is at least one gene selected from the group consisting of CD55, HMOX1, TSPAN7, RAB27B, IL, GPX3, and MFAP4, preferably at least 2, at least 3, or at least 4 of these genes, more preferably at least 5, at least 6, or at least 7 of these genes, and particularly preferably all of these genes are enhanced.

The expression level of the specific gene in the mesenchymal stem cell of the present invention may be generally increased by 1.1-fold or more, preferably by 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 4.5-fold or more, 5.0-fold or more, 5.5-fold or more, 6.0-fold or more, 6.5-fold or more, 7.0-fold or more, 7.5-fold or more, 8.0-fold or more, 8.5-fold or more, 9.0-fold or 9.5-fold or 10.0-fold or more, as compared to the expression level of the specific gene in the mesenchymal stem cell cultured by the adherent culture as a control.

The culture conditions of the mesenchymal stem cells used as a control are not particularly limited as long as they can be maintained and/or cultured by proliferation under the conditions of adhesion. As an example, the conditions used in the examples of the present application (medium: mesenchymal stem cell proliferation medium 2 (PromoCell Co., # C-28009), vessel: 10cm dish (Corning Co., # 430167), temperature: 37 ℃ C., CO ₂ concentration: 5%) are mentioned, but not limited thereto.

In one embodiment, the condition for the adherent culture of the mesenchymal stem cells is two-dimensional culture using a culture dish.

Whether or not the expression of these genes is elevated can be determined by a method known per se. For example, as shown in the embodiments described later, a method using real-time PCR may be exemplified, but is not limited thereto.

In a preferred embodiment of the mesenchymal stem cell of the present invention, the mesenchymal stem cell of the present invention is characterized in that the expression level of at least 1 protein selected from the group consisting of PGE2, RAB27B, NFE L2 (or also referred to as "NRF 2"), P65 and P-P65 (phosphorylated form of P65) is increased. In one embodiment, the mesenchymal stem cells of the present invention have an increased expression level of any 2 of PGE2, RAB27B, NFE L2, P65 and P-P65. In one embodiment, the mesenchymal stem cells of the present invention have an increased expression level of any 3 of PGE2, RAB27B, NFE L2, P65 and P-P65. In one embodiment, the mesenchymal stem cells of the present invention have an increased expression level of any 4 of PGE2, RAB27B, NFE L2, P65, and P-P65. In one embodiment, the mesenchymal stem cells of the present invention have increased expression levels of all of PGE2, RAB27B, NFE L2, P65 and P-P65.

The protein expression level of RAB27B, NFE L2, P65 and/or P-P65 in the mesenchymal stem cells of the present invention can be usually 1.1-fold or more, preferably 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 4.5-fold or more, 5.0-fold or more, 5.5-fold or more, 6.0-fold or more, 7.0-fold or more, 7.5-fold or more, 8.0-fold or more, 8.5-fold or more, 9.0-fold or more, or not, compared with the protein expression level in the mesenchymal stem cells cultured by the adherent culture as a control.

Whether or not the expression level of these proteins is increased can be determined by a method known per se. For example, a method using Western blotting and a method using ELISA can be exemplified, but not limited thereto.

The tissue from which the mesenchymal stem cells of the present invention are derived is not particularly limited, and may be a mesenchymal stem cell derived from any tissue. For example, the mesenchymal stem cells of the present invention may be derived from umbilical cord, bone marrow, adipose tissue, or peripheral blood. The mesenchymal stem cells of the present invention may preferably be derived from umbilical cord, bone marrow or adipose tissue, more preferably may be derived from umbilical cord or adipose tissue, and particularly preferably may be derived from adipose tissue.

In addition, the mesenchymal stem cells of the present invention promote the production of extracellular vesicles as compared to mesenchymal stem cells cultured by adherent culture.

Extracellular vesicles (extracellular vesicle: EV) are small cells formed from lipid bilayer membranes. Based on the differences in the mechanism of formation, extracellular vesicles are largely classified as exosomes, microvesicles, and apoptotic bodies. In one embodiment of the invention, the extracellular vesicles are exosomes.

The exosomes contain "cargo" (e.g., mRNA, miRNA, protein, lipid, etc.), and it is known that the amount and type of these cargo varies depending on the state of the cells secreting the exosomes. Therefore, development of detection techniques for diseases based on analysis of exosomes and development of therapeutic methods for diseases using exosomes as therapeutic targets are advancing.

Exosomes are reported to be secreted by various kinds of cells, in particular exosomes secreted by mesenchymal stem cells are reported to have very interesting properties. Mesenchymal stem cells have an ability to differentiate into various cells, and moreover, have a low risk of forming tumors, and for this reason, their use in regenerative medicine is being developed. Here, it was shown that the therapeutic effect resulting from the transplantation of mesenchymal stem cells depends on body fluid factors such as mRNA, miRNA, protein, and lipid contained in exosomes derived from the transplanted mesenchymal stem cells (Spees JL et al STEM CELL RES Ther.2016Aug 31;7 (1): 125.). Therefore, research using exosomes derived from mesenchymal stem cells as therapeutic agents has also progressed, and it has been reported that exosomes derived from mesenchymal stem cells inhibit fibrosis of tissues in liver diseases and kidney diseases (Kan Yin et al Biomark res.2019apr4; 7:8), and have therapeutic effects in heart diseases, alzheimer diseases, and the like (Matthew H Forsberg et al, front Cell Dev biol.2020jul 17; 8:665.). Therefore, the mesenchymal stem cells of the present invention, in which the ability to produce exosomes is promoted, have the possibility of being useful as a therapeutic or prophylactic drug for various diseases.

In one embodiment, the amount of extracellular vesicles produced in the mesenchymal stem cells of the present invention may be generally 1.1-fold or more, preferably 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 4.5-fold or more, 5.0-fold or more, 5.5-fold or more, 6.0-fold or more, 6.5-fold or more, 7.0-fold or more, 7.5-fold or more, 8.0-fold or more, 8.5-fold or more, 9.0-fold or 9.5-fold or 10.0-fold or more, as compared to the amount of extracellular vesicles produced in the mesenchymal stem cells cultured by the adherent culture as a control.

In another aspect, the amount of exosomes produced in the mesenchymal stem cells of the present invention may be generally 1.1-fold or more, preferably 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 3.5-fold or more, 4.0-fold or more, 4.5-fold or more, 5.0-fold or more, 5.5-fold or more, 6.0-fold or more, 6.5-fold or more, 7.0-fold or more, 7.5-fold or more, 8.0-fold or more, 8.5-fold or 9.0-fold or more, 9.0-fold or 10.0-fold or more, as compared to the amount of exosomes produced in the mesenchymal stem cells cultured by the adherent culture as a control.

In addition, as shown in the examples below, the mesenchymal stem cells of the present invention are characterized by an increased production of "PGE2" as compared to mesenchymal stem cells produced by adherent culture. PGE2 is known to be one of secreted proteins with anti-inflammatory effects. Therefore, the mesenchymal stem cells of the present invention can be suitably used as an anti-inflammatory agent.

6. Method for promoting production of extracellular vesicles of mesenchymal stem cells

The present invention also provides a method for promoting the production of extracellular vesicles of mesenchymal stem cells (hereinafter, sometimes referred to as "the method for promoting the production of mesenchymal stem cells") comprising the step of culturing mesenchymal stem cells in suspension in a medium comprising nanofibers composed of a water-insoluble polysaccharide, wherein the culturing is performed with stirring.

The implementation of the promotion method of the present invention is the same as that of culturing mesenchymal stem cells by the method of the present invention. Accordingly, the various conditions in the promotion method of the present invention are the same as those described in the method of the present invention. The mesenchymal stem cells of the present invention can be obtained by culturing the mesenchymal stem cells using the acceleration method of the present invention. The method of promoting the present invention may be referred to as a method for producing mesenchymal stem cells in which the production of extracellular vesicles is promoted.

7. Preparation for treating inflammatory diseases comprising mesenchymal stem cells of the present invention

The present invention also provides a preparation for treating an inflammatory disease (hereinafter, sometimes referred to as "therapeutic agent for an inflammatory disease of the present invention") comprising the mesenchymal stem cells of the present invention.

As described above, the mesenchymal stem cells of the present invention have an increased secretion of PGE2 having an anti-inflammatory effect. Therefore, the mesenchymal stem cells of the present invention are extremely useful as a therapeutic agent for inflammatory diseases.

The amount of the mesenchymal stem cells of the present invention contained in the therapeutic agent for inflammatory diseases of the present invention is not particularly limited, but is usually 0.001 wt% or more, preferably 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, or 0.5 wt% or more, and more preferably 1 wt% or more, based on the weight of the entire preparation. The upper limit is not particularly limited, and may be generally 100 wt% or less, preferably 90 wt% or less, 70 wt% or less, 50 wt% or less, or 30 wt% or less, and more preferably 10 wt% or less. In one embodiment, the amount of the mesenchymal stem cells of the present invention contained in the anti-inflammatory agent of the present invention is usually 0.001 to 100 wt%, preferably 0.01 to 90 wt%, 0.05 to 70 wt%, 0.1 to 50 wt% or 0.5 to 30 wt%, more preferably 1 to 10 wt%, but not limited thereto.

The therapeutic agent for inflammatory diseases of the present invention may contain other components than the mesenchymal stem cells of the present invention. As the other ingredient, for example, a pharmaceutically acceptable pharmaceutical additive may be contained. Examples of the pharmaceutical additives include, but are not limited to, isotonic agents, buffers, pH adjusting agents, stabilizers, chelating agents, preservatives, and the like.

Examples of isotonic agents include sodium chloride, potassium chloride, saccharides, glycerol, and the like. Examples of the buffer include boric acid, phosphoric acid, acetic acid, citric acid, and salts thereof (e.g., alkali metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, and alkaline earth metal salts thereof). The pH regulator may be inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, polyphosphoric acid, boric acid, or borax; organic acids such as acetic acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, citric acid, succinic acid, tartaric acid, and malic acid; inorganic bases such as potassium hydroxide and sodium hydroxide; organic bases such as monoethanolamine, triethanolamine, diisopropanolamine, and triisopropanolamine; ammonium acetate, sodium lactate, sodium citrate, potassium carbonate, sodium bicarbonate, sodium carbonate, ammonium bicarbonate, dipotassium phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, calcium lactate, etc. Examples of the stabilizer include human serum albumin, usual L-amino acids, saccharides, cellulose derivatives, etc., which may be used alone or in combination with a surfactant, etc. The L-amino acid may be any of glycine, cysteine, glutamic acid, etc., but is not limited thereto. The saccharide may be any of monosaccharides such as glucose, mannose, galactose, fructose, sugar alcohols such as mannitol, inositol, xylitol, disaccharides such as sucrose, maltose, lactose, polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, and the like, and derivatives thereof, but is not limited thereto. The cellulose derivative may be any of methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, etc., but is not limited thereto. Chelating agents may be exemplified by sodium ethylenediamine tetraacetate, citric acid, and the like.

The shape of the therapeutic agent for inflammatory diseases of the present invention is not particularly limited as long as it can be administered to a subject parenterally. For example, it may be made into a liquid form containing cells and a suitable dispersion medium. In addition, in the case where the therapeutic agent for inflammatory diseases of the present invention is directly applied to a severe part or the like, the therapeutic agent for inflammatory diseases of the present invention may be formed into a sheet shape obtained by fixing mesenchymal stem cells to a biocompatible material.

The amount of the therapeutic agent for inflammatory diseases of the present invention to be administered to a subject is not particularly limited, and may be any amount as long as it can reduce inflammatory reaction. The amount may be appropriately determined in consideration of the degree of inflammation, the age, weight, administration method, the number of administration times, the shape of the therapeutic agent for inflammatory diseases of the present invention, and the like of the subject.

In one embodiment, the therapeutic agent for inflammatory diseases of the present invention is applied to a subject suffering from inflammatory diseases. As a result of the inflammatory disease(s), inflammatory bowel disease, ulcerative colitis, crohn's disease, nephritis, acute nephritis, chronic nephritis, glomerulonephritis, igA nephropathy, diabetic nephropathy, membranous nephropathy, hydronephrosis, contrast nephropathy, pyelonephritis, renal failure, interstitial nephritis, kidney injury, nephrotic syndrome, hypertensive nephrosclerosis, diabetic glomerulosclerosis, kidney stones, amyloidosis nephropathy, renal venous thrombosis, alport syndrome, hepatitis, cirrhosis, pancreatitis, pneumonia, sinusitis, rhinitis, arthritis (arthrosis), deformable knee joint disease, deformable hand joint disease, deformable foot joint disease, deformable hip joint disease, rheumatoid arthritis, periodic fever, aphtha pharyngitis lymphadenitis syndrome (PFAPA), adult Steve disease Behcet's disease, gout, pseudogout, schnitzler syndrome, chronic Recurrent Multifocal Osteomyelitis (CRMO), cold porphyrin-related cyclic fever syndrome (CAPS), familial cold urticaria, muckle-Wells syndrome, chronic infant neurodermal arthritis syndrome (CINCA syndrome)/neonatally ill multisystemic inflammatory disease (NOMID), TNF (tumor necrosis factor) receptor-related cyclic syndrome (TRAPS), high IgD syndrome (mevalonate kinase deficiency), blau syndrome/early-onset sarcoidosis, familial mediterranean fever, PAPA (suppurative arthritis-gangrene-necrosis-acne) syndrome, mid-Welch syndrome, majeed syndrome, NLRP 12-related cyclic fever syndrome (NAPS 12), interleukin 1 receptor antagonist Deficiency (DIRA), interleukin 36 receptor antagonist Deficiency (DITRA), phospholipase C gamma 2 related antibody deficiency, immune disorder (PLAID), HOIL-1 deficiency, SLC29A3 deficiency, CARD14 abnormality, ADA2 (adenosine deaminase 2) deficiency, STING related infantile vascular diseases (SAVI) and NLRC4 abnormality, but are not limited thereto. In one embodiment, a suitable disease for use as an anti-inflammatory agent of the present invention is arthropathy, and in particular, may be deformable knee arthropathy, deformable hand arthropathy, deformable foot arthropathy, or deformable hip arthropathy.

The target to which the therapeutic agent for inflammatory diseases of the present invention is applied is not particularly limited as long as it is a living organism likely to suffer from inflammatory diseases, and is usually a mammal such as a rat, a mouse, a rabbit, a guinea pig, a squirrel, a hamster, a field mouse, a duckbill, a dolphin, a whale, a dog, a cat, a goat, a cow, a horse, a sheep, a pig, a marmoset, a squirrel, a rhesus monkey, a chimpanzee, or a human, and preferably a human.

The mesenchymal stem cells of the present invention contained in the anti-inflammatory agent of the present invention may be spherical, or may be single-celled cells, or may be a mixture of these. In one embodiment, the mesenchymal stem cells of the present invention contained in the anti-inflammatory agent of the present invention may be single-celled cells.

The anti-inflammatory agent of the present invention can be administered to a subject, thereby treating inflammatory diseases in the subject. Accordingly, the present invention provides methods for treating an inflammatory disease in a subject.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Examples

Preparation example 1 preparation of a substrate

An aqueous dispersion comprising vitronectin-supported chitin nanofibers and chitosan nanofibers was prepared according to the descriptions of WO2015/111686 and WO 2021/002448. Specifically, the preparation was performed as follows. An aqueous dispersion of 2 mass% chitin nanofibers prepared as described in WO2015/111686 was autoclaved at 121℃for 20 minutes. Then, this aqueous dispersion was mixed and suspended in sterile distilled water (tsukamurella distilled water, manufactured by tsukamurella pharmaceutical factory, ltd.) so as to be 1% (w/v), to prepare a sterile chitin nanofiber-containing aqueous dispersion. To 1% (w/v) of an aqueous dispersion of chitin nanofibers (5 mL), an aqueous dispersion containing vitronectin-loaded chitin nanofibers was prepared by adding an aqueous solution (Gibco Vitronectin (VTN-N) recombinant human protein, manufactured by Thermo FISHER SCIENTIFIC company) (0.5 mL) containing 500. Mu.g/mL, mixing the aqueous solution by blowing, and then allowing the mixture to stand at 4℃for storage overnight. As a result of the analysis according to WO2021/002448, the amount of vitronectin supported was 20. Mu.g/mL (2.2 mg per 1g of chitin nanofiber). Next, the 2 mass% aqueous dispersion of chitosan nanofibers prepared as described in WO2015/111686 was subjected to autoclave sterilization at 121℃for 20 minutes. Then, the aqueous dispersion was mixed and suspended in sterile distilled water (tsukamurella distilled water, manufactured by tsukamurella pharmaceutical factory, ltd.) so as to be 1% (w/v), whereby a sterile aqueous dispersion containing chitosan nanofibers was produced. The prepared aqueous dispersion of chitosan nanofibers (8 mL) was added to the above aqueous dispersion of chitin nanofibers containing vitronectin (2 mL) and mixed by blowing, thereby preparing an aqueous dispersion of chitin nanofibers containing vitronectin (0.2% (w/v) and chitosan nanofibers (0.8% (w/v)) containing (10 mL). (in this specification, the mixture of vitronectin-supported chitin nanofibers and chitosan nanofibers prepared herein is sometimes referred to simply as "substrate of preparation example 1", "preparation example 1" or "substrate 1")

Test example 1 culture with stirring 1 using substrate 1 (comparison with microcarriers)

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a10 cm dish (Corning, co., # 430167) for 3 days. Cells were then detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), added to 15mL of medium so as to have an inoculation concentration of 3X 10 ⁴ cells/mL, and cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under various conditions for 10 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a special magnetic stirrer (manufactured by ZZABLE Corporation, # BWS-S03N 0S-6) was used under stirring conditions. On days 4 and 7 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange.

(Culture conditions)

In comparative example 1, corning (registered trademark) Low Concentration Synthemax (registered trademark) II Microcarriers (manufactured by Corning corporation, # 3781) was weighed, immersed in ethanol for sterilization (manufactured by also pharmaceutical industry corporation, # 4987556241025), and then replaced with mesenchymal stem cell growth medium 2, and cultured under stirring using a microcarrier corresponding to 300 mg. For stirring, the culture was carried out at 55rpm for 1 minute and at 0rpm for 59 minutes as1 cycle, and after 10 cycles of culture under this condition, continuous stirring was carried out at 55 rpm. In examples 1 and 2, a culture medium composition was used in which the substrate of preparation example 1 was added to the mesenchymal stem cell growth medium 2 so as to have a final concentration of 0.05% (w/v). In addition, example 1 was cultured under static conditions, and example 2 was cultured under stirring conditions similar to those of comparative example 1.

(Calculation of proliferation Rate)

On days 0, 1, 4, 7 and 10 of the culture, 0.5mL of the uniformly suspended culture solution was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added thereto, stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 150. Mu.L of each of the solution was dispensed into a white 96-well plate, luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 1.

TABLE 1

	Day 0	Day 1	Day 4	Day 7	Day 10
						Comparative example 1	1	1.4	12.7	14.9	12.3
Example 1	1	0.7	3.6	15.3	10.1
						Example 2	1	0.7	2.8	8.7	14.4

(Determination of Medium Environment)

On days 0, 1, 4, 7 and 10 of the culture, 0.5mL of the culture medium in uniform suspension was collected in a 1.5mL tube, and the culture supernatant was obtained by centrifugation (600 Xg, 3 minutes). Glucose, lactic acid and ammonia concentrations in the culture supernatants were measured using FLEX2 (nova biomedical Co.). As a measurement value on day 0, a medium in a cell-free state before the start of culture was used for measurement. The results are shown in Table 2.

TABLE 2

Glucose (mM)	Day 0	Day 1	Day 4	Day 7	Day 10
						Comparative example 1	4.6	4.2	0.9	0.0	0.0
Example 1	4.6	4.3	3.2	0.0	0.0
						Example 2	4.6	4.2	3.2	0.0	0.0

						Lactic acid (mM)	Day 0	Day 1	Day 4	Day 7	Day 10
Comparative example 1	1.1	3.7	8.1	8.1	7.5
						Example 1	1.1	3.9	3.4	9.5	9.7
Example 2	1.1	3.4	3.1	9.0	8.4

Ammonia (mM)	Day 0	Day 1	Day 4	Day 7	Day 10
						Comparative example 1	0.11	0.15	1.00	2.46	2.43
Example 1	0.11	0.16	0.63	1.28	1.54
						Example 2	0.11	0.16	0.68	1.39	1.93

As shown in table 1, the proliferation rate of comparative example 1 increased sharply on day 4 and reached maximum on day 7. On the other hand, the proliferation rates of examples 1 and 2 increased sharply after day 4, with example 1 reaching a maximum on day 7 and example 2 reaching a maximum on day 10. The maximum values are substantially the same. From the above results, it was revealed that the maximum value of the proliferation rate was not different between the conditions of the culture in which the medium composition used in the example was allowed to stand or stirred without interruption and the conditions of comparative example 1.

As shown in table 2, the ammonia concentration in the culture media of examples 1 and 2 was always lower than that of comparative example 1. In addition, the glucose concentration in the medium was substantially 0 on day 7 under any conditions, but the proliferation rate of example 2 increased to day 10 with time. From the above results, it was revealed that by using the medium composition used in the examples, cultivation with reduced ammonia having cytotoxicity can be achieved, and cells can proliferate even at a low glucose concentration.

Test example 2 culture with stirring 2 (stirring conditions and CO ₂ concentration) using substrate 1

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Cells were then detached using DETACHKIT (manufactured by PromoCell, #C-41210) and added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 at a final concentration of 0.05% (w/v) so as to achieve an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃, 5% or 10% CO ₂) under various conditions for 7 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a special magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used under stirring conditions. The culture vessel was allowed to stand for 10 minutes on day 4 of the culture, and half of the culture supernatant was subjected to medium exchange.

(Culture conditions)

Stirring and CO ₂ conditions are shown below.

Condition 1: standing, 5% CO ₂

Condition 2: standing, 10% CO ₂

Condition 3: stirring at 25rpm without interruption, 5% CO ₂

(Calculation of proliferation Rate)

On days 0, 1, 4 and 7 of the culture, 0.5mL of the uniformly suspended culture medium was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added to each, stirred by a vortex mixer, left standing at room temperature for 10 minutes, and 150. Mu.L of each of the white 96-well plates was dispensed, and luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation) and the luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 3.

TABLE 3

	Day 0	Day 1	Day 4	Day 7
					Condition 1	1.0	1.4	5.6	7.7
Condition 2	1.0	1.3	3.8	4.5
					Condition 3	1.0	1.5	10.5	14.3

As shown in table 3, the proliferation rate was reduced under condition 2 as compared with condition 1. In addition, the proliferation rate was improved under condition 3as compared with condition 1. From the above results, it was found that the stirring frequency during culture can affect the proliferation efficiency. In addition, comparison with stationary culture shows that the proliferation efficiency can be improved under continuous stirring culture.

Test example 3 culture with stirring 3 using substrate 1 (comparison with stationary culture)

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell growth medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃, 5% CO ₂) for 9 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and in terms of stirring conditions, a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used to perform continuous stirring at 25 rpm. On days 4 and 7 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange. In addition, as a comparison object, cells were inoculated into a 24-well flat bottom adhesion surface microplate (# 3526, manufactured by corning corporation) so as to be 5×10 ⁴ cells/well/1 mL, and adhesion culture was performed.

(Culture conditions)

Condition 4: standing still

Condition 5: uninterrupted stirring

(Calculation of proliferation Rate)

On days 0,1,2,3, 4, 7, 8, and 9 of the culture, 0.5mL of the uniformly suspended culture solution was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added to each, the mixture was stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 150. Mu.L of each was dispensed to a white 96-well plate, and the luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and the luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value (ATP measurement, luminescence intensity) on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 4.

(Analysis of Gene expression)

Cells were collected on days 0, 4 and 7 of culture, 700. Mu.L of RLT solution (RNEASY MINI KIT (manufactured by QIAGEN, # 74106) was added to the RNA extraction solution, and then, 70% ethanol was added to the RNeasy centrifugation column, followed by centrifugation at 8000 Xg for 15 seconds, 700. Mu.L of RW1 solution was added to the RNeasy centrifugation column, centrifugation at 8000 Xg for 15 seconds, 500. Mu.L of RPE solution was then added, 500. Mu.L of RPE solution was further added, centrifugation at 8000 Xg for 2 minutes, no RNase solution was added to RNA present in the RNeasy centrifugation column, and elution was performed, then, cDNA was synthesized from the obtained RNA using PRIMESCRIPT RT REAGENT KIT (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 037A) using synthesized cDNA and Premix Taq (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # 039A) and Takara Inc probe Applied Bio Systems) as PCR values, and Hs were calibrated by using a PCR apparatus having a value of GHs 1, such as that 20 Hs 4 were calibrated by using a PCR device (manufactured by using GHs 4, hs 1 to 35, hs 4, and Hs 4 were calibrated by using a Real-Time scale of GHs 4.

(Cell staining)

On day 7 of the culture, 1mL of the culture medium in uniform suspension was collected into a 1.5mL tube, and after centrifugation (600 Xg, 3 minutes), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (600 Xg, 3 minutes). 10. Mu.L of a solution of Calcein-AM (manufactured by Tokugaku Co., # C326) dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 5mL of D-PBS (-) (manufactured by Fuji photo-pure chemical Co., # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 30 minutes. Then, using EVOS (registered trademark) FL Auto (manufactured by ThermoFisher Co.) a bright field image and a living cell-specific fluorescent staining image were obtained. The results are shown in FIG. 1. The scale bar represents 1000. Mu.m.

(Image analysis)

Image analysis was performed using image J (National Institutes of Health, 64-bit Java 1.8.0-172) using fluorescent staining images obtained by cell staining. As a pre-process of image analysis, implementation: normalization of the scale in the image is used; 32-bitification of each image; unifying brightness among the images; adaptation of Gaussian filter (Sigma value 2.00); contour extraction of FIND EDGES was used; adaptation of the Binary process and Close; and removing the image overlapped with the image edge by the scale in the image. Using the image obtained by the pretreatment, spheres having an area value of 17671.46 (μm ²) or more (average diameter of 150 μm or more) were extracted, and the number of spheres, the area value (μm ²), and the roundness were obtained. Using the obtained area value, the average diameter of the sphere when the sphere is assumed to be a perfect circle is calculated, and the average diameter of the sphere, the standard deviation, and the size distribution data are obtained. The average diameter of the sphere is calculated using the following formula. The sphere-extracted image of which data was finally obtained is shown in fig. 2, the number of spheres, the average diameter and standard deviation of spheres, the roundness and standard deviation are shown in table 6, and the sphere size distribution is shown in fig. 3. The X-axis in FIG. 3 represents, for example, 150 to 175, the number of spherical groups of 150 μm or more and less than 175 μm.

[ Mathematics 1]

TABLE 4

	Day 0	Day 1	Day 2	Day 3	Day 4	Day 7	Day 8	Day 9
									Condition 4	1	1.3	2.1	2.9	4	9	9.5	7.9
Condition 5	1	1.3	2.2	3.7	6.9	12.2	16.6	15.5

As shown in table 4, a high proliferation rate was obtained under condition 5 as compared with condition 4.

TABLE 5

As shown in table 5, under conditions 4 and 5, the relative gene expression amounts of OCT4, nanog, CXCR4 increased over time as compared to the cells at the time of inoculation. Further, condition 5 is a higher value than condition 4.

TABLE 6

As shown in fig. 3, under condition 4, the number of clusters of 150 μm or more and less than 175 μm was the largest, and no clear peak top was observed, but under condition 5, the number of clusters of 250 μm or more and less than 275 μm was the largest, and a bell-like distribution was obtained. As shown in table 6, the standard deviation under condition 5 was smaller and the roundness was higher than that under condition 4. From the above results, it was revealed that by stirring, a more uniform spherical body could be obtained.

Test example 4 culture with stirring 4 (substrate concentration) using substrate 1

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to the mesenchymal stem cell growth medium 2 at a final concentration of 0.05% (w/v), 0.02% (w/v) or 0.01% (w/v) so as to achieve an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing the cells in a CO ₂ incubator (37 ℃ C., 5% CO ₂) with stirring for 7 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and stirring was performed using a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) with no break at 25 rpm. The culture vessel was allowed to stand for 10 minutes on day 4 of the culture, and half of the culture supernatant was subjected to medium exchange.

(Calculation of proliferation Rate)

On days 0, 1,2, 4 and 7 of the culture, 0.5mL of the uniformly suspended culture solution was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added to each, the mixture was stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 150. Mu.L of each was dispensed to a white 96-well plate, luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value (ATP measurement, luminescence intensity) on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 7.

(Cell staining)

On day 7 of the culture, 1mL of the culture medium in uniform suspension was collected into a 1.5mL tube, and after centrifugation (600 Xg, 3 minutes), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (600 Xg, 3 minutes). 10. Mu.L of a solution of Calcein-AM (manufactured by Tokugaku Co., # C326) dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 5mL of D-PBS (-) (manufactured by Fuji photo-pure chemical Co., # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 30 minutes. Then, using EVOS (registered trademark) FL Auto (manufactured by ThermoFisher Co.) a bright field image and a living cell-specific fluorescent staining image were obtained. A representative image is shown in fig. 4. The scale bar represents 1000. Mu.m.

(Image acquisition)

On day 7 of the culture, 0.5mL of the uniformly suspended culture solution was collected in a 12-well plate, and the whole of the well was photographed using Cell3iMagerduos (sceen holders co., ltd.). The acquired image is shown in fig. 5.

(Image analysis)

Image analysis using ImageJ (National Institutes of Health,64-bit Java 1.8.0 _172) was performed using the acquired image of the entire well. After the scale in the image was used to normalize the scale, the contour of the sphere was extracted using Polygon selections tool, and the number of spheres and the area value (μm ²) were obtained. Using the obtained area value, the average diameter of the sphere when the sphere is assumed to be a perfect circle is calculated, and the average diameter of the sphere is obtained. The average diameter of the sphere is calculated using the following formula. Fig. 6 shows an extracted sphere image of the data obtained finally, and fig. 7 shows the number of spheres and the average diameter of spheres. When extracting the contours, bubbles are excluded from the object.

[ Math figure 2]

TABLE 7

	Day 0	Day 1	Day 2	Day 4	Day 7
						0.05％	1.0	1.6	2.3	5.5	15.1
0.02％	1.0	1.6	2.7	5.7	8.4
						0.01％	1.0	1.7	2.4	4.4	6.4

As shown in table 7 and fig. 7, the present invention can be implemented even when the concentration of the base material 1 is changed.

Test example 5 separation of spheres

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 25rpm for 4 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used.

(Separation of spheres)

On the 3 rd day of culture, the whole amount of the culture solution was passed through a cell sieve (pluriSelect Co., # 43-50060-03) having a pore size of 60 μm, and then the net was turned upside down, and the medium was washed with an equal amount of mesenchymal stem cell proliferation medium 2, whereby the spheres trapped on the net were collected as a sphere suspension. The solution passing through the mesh was used as filtrate.

(Microscopic observation)

The uniformly suspended culture solution, the spherical suspension, and 0.5mL of the filtrate were transferred from the cell screen to a 12-well plate (manufactured by Corning Co., # 351143), and observed with an inverted microscope (manufactured by Olympus Corporation, # IX 73). The culture solution obtained by further culturing the spherical suspension for 1 day and the suspension obtained by culturing the spherical suspension for 4 days without treatment with the cell screen were similarly observed. The acquired image is shown in fig. 8. The scale bar represents 500. Mu.m.

(Dyeing)

The obtained filtrate (1 mL) was collected in a 1.5mL tube, and after centrifugation (600 Xg, 3 minutes), the supernatant was removed. The precipitate (pellet) was suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the supernatant was removed after centrifugation (600 Xg, 3 minutes). 10. Mu.L of a solution of Calcein-AM (manufactured by Tokugaku Co., # C326) dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 5mL of D-PBS (-) (manufactured by Fuji photo-pure chemical Co., # 045-29795) as a staining solution. The precipitate was suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 30 minutes. Then, using EVOS (registered trademark) FL Auto (manufactured by ThermoFisher Co.) a bright field image and a living cell-specific fluorescent staining image were obtained. The acquired image is shown in fig. 9. The scale bar represents 1000. Mu.m.

As shown in fig. 8 and 9, it was demonstrated that the spheres could be separated from the excess substrate by using a cell screen, and the culture was continued. From the above results, it was revealed that by using a net, only spheres can be separated from the substrate, single cells, and dead cells.

Test example 6 expansion culture

A medium containing mesenchymal stem cell proliferation medium 2 (manufactured by PromoCell Co., # C-28009) and penicillin-streptomycin solution (. Times.100) (manufactured by Fuji photo-Paul et al, # 168-23191) was added to UniVessel (registered trademark) Glass1L (manufactured by Sartorius Co., ltd.) sterilized (121 ℃ C., 20 minutes) by an autoclave, and the mixture was connected to BIOSTAT (registered trademark) B-DCU (manufactured by Sartorius Co., ltd.) to adjust the medium for 30 minutes under conditions of compressed air of 130ccm and CO ₂ of 10ccm, 37 ℃ C., 120 rpm. For mesenchymal stem cells derived from human umbilical cord (PromoCell, inc. # C-12971), mesenchymal stem cell proliferation medium 2 was used, and the culture was performed on a 10cm dish (Corning, inc. # 430167) for 3 days. Then, cells were removed by using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the resulting mixture was added to a medium in which the substrate of preparation example 1 was adjusted so that the final concentration was 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL. The total amount of medium was 424mL. The culture was performed under stirring at 37℃and 45 or 60rpm for 11 days with compressed air of 130ccm and CO ₂ of 8 or 10 ccm. After half of the cell suspension in the reactor was recovered and centrifuged (300×g,3 min, deccel mode) on days 4 and 7 of the culture, the supernatant was removed, suspended in a new medium, and returned to the reactor, whereby the medium was replaced.

(Calculation of proliferation Rate)

On days 0, 1,4, 7 and 11 of the culture, 0.5mL of the uniformly suspended culture solution was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added thereto, the mixture was stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 150. Mu.L of each solution was dispensed into a white 96-well plate, luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and the luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value (ATP measurement, luminescence intensity) on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 8.

(Separation of spheres)

On day 11 of the culture, 50mL of the culture solution was passed through a cell sieve having a pore diameter of 100 μm (pluriSelect, manufactured by the company # 43-50100-03) or a cell sieve having a pore diameter of 200 μm (pluriSelect, manufactured by the company # 43-50200-03), and then D-PBS (-) (Fuji film and Wako pure chemical industries, manufactured by the company # 045-29795) was added thereto, whereby the spheres captured on the mesh were washed, the mesh was turned upside down, and the spheres captured on the mesh were washed with an appropriate amount of D-PBS (-) to be collected as a sphere suspension. The solution passing through the mesh was used as filtrate.

(Microscopic observation)

1ML of the culture solution, the spherical suspension, and the filtrate before passing through the cell screen, which were uniformly suspended, were transferred to a 12-well plate (manufactured by Corning Co., # 351143), and observed using an inverted microscope (manufactured by Olympus Corporation, # IX 73). The acquired image is shown in fig. 10. The scale bar represents 500. Mu.m.

TABLE 8

	Day 0	Day 1	Day 4	Day 7	Day 11
						Proliferation rate	1.0	1.5	2.8	6.4	12.2

As shown in table 8, it was revealed that cells proliferated with time even under the expanding conditions. In addition, the stirring blade of the bioreactor manufactured by Sartorius Corporation used in this example was in the shape of a screw, and the stirring blade of the bioreactor manufactured by ABLE Corporation used in the previous example was in the shape of a triangle (delta-type), thus indicating that the culture can be expanded regardless of the shape of the stirring blade.

As shown in fig. 10, it was clarified that the spheres can be separated from the excessive substrate by using a cell screen. In addition, in the case of using a cell sieve having a pore size of 100 μm, a substrate having a smaller pore diameter than the mesh diameter cannot be completely washed due to clogging, but by using a cell sieve having a pore size of 200 μm, clogging is eliminated, and there are few cases where a small substrate is mixed into the sphere suspension after washing. From the above results, it was found that by selecting a cell screen with an appropriate pore size for recovery and washing of spheres, improvement of washing efficiency and increase of the amount of liquid that can be treated can be expected.

Test example 7 Single-cell formation of spheres

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 100mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell growth medium 2at a final concentration of 0.05% (w/v) so as to have an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃, 5% CO ₂) for 10 days. As a culture vessel, a 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A) was used, and continuous stirring was performed at 25rpm using a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6). On the 3 rd day of culture, the whole amount of the culture solution was passed through a cell sieve (pluriSelect, inc. # 43-50060-03) having a pore size of 60 μm, and then the net was turned upside down, and the culture was continued by washing with an equal amount of mesenchymal stem cell proliferation medium 2, whereby the spheres trapped on the net were collected. On day 7 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange.

(Enzyme treatment)

To 2 vials of Dri Tumor & Tissue Dissociation Reagent (TTDR) (BD Co., # 661563) were added 5mL of D-MEM medium (Fuji photo-pure Co., # 043-30085) and the mixture was dissolved at room temperature for 15 minutes while being properly mixed. To a total of 10mL TTDR of the solution, 2mL of TrypLE (registered trademark) Select Enzyme (10X) phenol red-free (manufactured by Thermo Fisher Co., # A1217701) was added as an Enzyme solution. 60mL of the culture medium uniformly suspended was collected into 250 mL tubes, and after centrifugation (300 Xg, 3 minutes), the culture supernatant was removed. The precipitate was suspended in 50mL of D-MEM medium, collected in 1 tube, and centrifuged (300 Xg, 3 minutes), and the supernatant was removed. The precipitate was suspended in an appropriate amount of D-MEM medium, combined with the enzyme solution to adjust to 20mL, transferred to a 100mL disposable reactor, and stirred in a CO ₂ incubator (37 ℃ C., 5% CO ₂) at 25rpm for 30 minutes using a special magnetic stirrer. Then, 30mL of a D-MEM medium containing 2% FBS was added to the reactor, and the cell suspension was transferred to a process bag (process bag).

(Separation)

The drain bag, the buffer bag containing the D-MEM medium containing 2% FBS, the intermediate storage bag, the bag containing the cell suspension, and the cell collection syringe were connected to Rotea a disposable line Kit (manufactured by Thermo Fisher Co., #A45130), and a flow path was branched between the drain bag and the Kit, and a flow path connected to the intermediate storage bag was added. A schematic illustration of the connection pattern is shown in fig. 11. Then, the cells were placed on Rotea (manufactured by Thermo Fisher Co.) and subjected to a dispersion treatment of spheres and cell separation. In the case of the set flow path, each step is performed after removing bubbles in the flow path by means of a vapor-water common (purge). In the sphere dispersion step, the centrifugal strength is changed to 100×g or 2000×g at 110mL/min, and the formation of the bet (bet formation) and the disintegration of the formed bet are repeated 10 times, whereby the spheres are dispersed into single cells. In the separation step, the 1 st stage was carried out at 110mL/min under 800 Xg conditions, and the 2 nd stage was carried out at 50mL/min under 2500 Xg conditions. In each washing step, the formed bet was washed with 50mL of buffer. In the cell recovery step, 20mL of the buffer was transferred to collect the cell fraction, which is the formed bet, into a syringe. The flow of the liquid in each step and the flow path used are shown in table 9.

(Purification)

4.5ML of Percoll (manufactured by Cytiva Co., # 17089101) and 0.5mL of 10 XD-PBS (-) (manufactured by Fuji film and Wako pure chemical industries, ltd., # 048-29805) were mixed with 4mL of a D-MEM medium containing 2% FBS in 4 15mL tubes, and a density gradient solution was prepared, and a density gradient was formed by centrifugation (400 Xg, 10min, slow Deccel mode). Then, 5mL of the cell fraction was slowly dispensed into each 15mL tube, and after centrifugation (400 Xg, 10min, slow Deccel mode), the cell fraction observed near the density gradient interface was collected into the 15mL tube, and the cells were concentrated by centrifugation (400 Xg, 3 min).

(Microscopic observation)

The culture medium of each stage was transferred to a 12-well plate (manufactured by Corning Co., # 351143) in 0.5mL or 1mL of the uniformly suspended state, and observed with an inverted microscope (manufactured by Olympus Corporation, # IX 73). In addition, the cell fraction was collected, purified, concentrated, and then stained specifically with trypan blue solution (Fuji photograph album and Wako pure chemical industries, ltd. # 207-17081) to obtain dead cells and a substrate, and then added to a counting plate (Bio-Rad Co. # 1450011) for observation in the same manner. The acquired image is shown in fig. 12. The scale bar represents 500. Mu.m.

TABLE 9

As shown in fig. 12, it is shown that spheres are dispersed into single cells after the dispersion step, and can be recovered as single cells with high purity by purification. From the above results, it was found that the formed spheres can be dispersed into single cells and recovered as single cells.

Test example 8 study 1 of the concentration of the substrate 1 and the recovery efficiency of cells

A culture medium composition was prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 (PromoCell Co., #C-28009) at a final concentration of 0.05 or 0.01% (w/v), respectively.

Next, the cultured mesenchymal stem cells (CellSource, manufactured by Corning Co., # 0111201) derived from human adipose tissue were each suspended in the above-described respective medium compositions so as to be 1.5X10- ⁴ cells/mL, and then inoculated into a 6-well flat-bottom ultra-low adhesion surface micro plate (manufactured by Corning Co., # 3471) at 10 mL/well. Cells were cultured in a CO ₂ incubator (37 ℃, 5% CO ₂) in a stationary state. The culture supernatant in the wells was removed by about 5mL on day 3, fresh 5mL of mesenchymal stem cell proliferation medium was added to each well, and suspended by a pipette, whereby half of the medium was replaced, and then further culture was continued until day 7 after inoculation. Half of the culture supernatant was removed on day 7 and the remainder was recovered into a 50mL conical tube. After allowing to stand for 15 minutes, the spheroids were naturally settled, and the remaining culture supernatant was removed. To this, HBSS (-) (manufactured by Thermo Fisher Co., # 14175095) (40 mL) was added, and the mixture was allowed to stand again for 15 minutes to allow the spheroid composition to naturally settle, and then the supernatant was removed and washed. Next, a liquid (100. Mu.L) in which Liberase (manufactured by Merck Co., # 5401119001) (5 mg) was dissolved in HBSS (-) (2 mL), TRYPLE SELECT Enzyme (10X), phenol red-free (manufactured by Thermo Fisher Co., # A1217701) (0.75 mL) and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 1 hour in a stationary state were added, and the cells were detached from the substrate. The obtained cell/substrate suspension (A) was filtered through a cell sieve (pluriSelect, inc. # 43-50100-51) having a mesh size of 100 μm, and the filtrate was washed with HBSS (-) (3 mL) to obtain a filtrate (B) containing cells separated from the substrate. Further, the filtrate was backwashed with HBSS (-) (10 mL) to obtain a filtrate suspension (C).

To 500. Mu.L of each of the above A, B, C, 500. Mu.L of ATP reagent (cell titer-Glo ^TM luminescence method cell viability assay, manufactured by Promega corporation) was added, suspended, allowed to stand at room temperature for about 10 minutes, and then dispensed into 3 wells at 300. Mu.L/well in a white 96-well plate, and the luminescence intensity (RLU value) was measured by an enzyme-labeled instrument (manufactured by Tecan corporation, INFINITEM200 PRO), and the luminescence value of the medium itself was subtracted to calculate the living cell amount (average value of 3 wells). Further, the volume of each suspension was divided, and the resultant ATP value was converted. The converted RLU values (ATP measurement, luminescence intensity) of the respective suspensions are shown in table 10.

TABLE 10

	0.05％	0.01％
			A) After peeling off	1648419	866728
B) Crude recovery	1303888	768530.1
			C) Residues of	347153.6	56077.41
B/A×100(％)	79.1	88.7
			C/A×100(％)	21.1	6.5

As is clear from table 10, it was confirmed that the amount of cells remaining on the substrate side was smaller in the case of 0.01% than in the case of 0.05% of the substrate 1. From this, it was found that the recovery efficiency of cells (single cells) was improved by reducing the amount of the substrate 1.

Test example 9 study 2 of the concentration of the substrate 1 and the recovery efficiency of cells

A culture medium composition was prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 (PromoCell Co., #C-28009) so that the final concentration became 0.100% (w/v), 0.050% (w/v), 0.020% (w/v), 0.0.10% (w/v), 0.005% (w/v) or 0.003% (w/v), respectively.

Next, the cultured mesenchymal stem cells (CellSource, manufactured by Corning Co., # 0111201) derived from human adipose tissue were each suspended in the above-described respective medium compositions so as to be 1.5X10- ⁴ cells/mL, and then inoculated into a 6-well flat-bottom ultra-low adhesion surface micro plate (manufactured by Corning Co., # 3471) at 10 mL/well. Cells were cultured in a CO ₂ incubator (37 ℃,5% CO ₂) in a stationary state. About 5mL of the culture supernatant in the well was removed on day 3, 5mL of fresh mesenchymal stem cell proliferation medium was added to each well, and the mixture was suspended by a pipette, whereby half of the culture medium was replaced, and then further culture was continued until day 7 after inoculation. 10mL of total volume was recovered from each well into a 15mL conical tube on day 7. After allowing to stand for 15 minutes, the spheroid composition was allowed to naturally settle, and then the remaining culture supernatant was removed. To this, HBSS (-) (manufactured by Thermo Fisher Co., # 14175095) (8 mL) was added, and the mixture was allowed to stand again for 15 minutes to allow the spheroid composition to naturally settle, and then the supernatant was removed and washed. Next, a liquid (80. Mu.L) in which Liberase (manufactured by Merck Co., # 5401119001) (5 mg) and TRYPLE SELECT Enzyme (10X) were dissolved in HBSS (-) (2 mL), phenol red-free (manufactured by Thermo Fisher Co., # A1217701) (300. Mu.L) were added, and the mixture was incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 1 hour in a stationary state, whereby cells were detached from the substrate. 5mL of the liquid (5.5 mL) obtained by diluting the obtained cell/substrate suspension (A) with HBSS (-) (4.1 mL) was filtered through a cell sieve (pluriSelect, inc. # 43-50070-51) having a mesh size of 70 μm, whereby a filtrate (B) (5 mL) containing cells separated from the substrate was obtained. Further, the filtrate was backwashed with HBSS (-) (5 mL) to obtain a filtrate suspension (C) (5 mL).

To 500. Mu.L of each of the above A, B, C, 500. Mu.L of ATP reagent (cell titer-Glo ^TM luminescence method cell viability assay, manufactured by Promega corporation) was added, suspended, allowed to stand at room temperature for about 10 minutes, and then, dispensed into 3 wells at 300. Mu.L/well of each of a white 96-well plate, luminescence intensity (RLU value) was measured by an enzyme-labeled instrument (manufactured by Tecan corporation, INFINITEM200 PRO), and the luminescence value of the medium itself was subtracted to calculate the living cell amount (average value of 3 wells). The converted RLU values (ATP measurement, luminescence intensity) of the suspensions and the ratio of the suspensions before and after the cell sieve was passed are shown in table 11.

TABLE 11

As is clear from table 11, it was confirmed that the recovery rate increased as the concentration of the base material was reduced. Further, although 0.010%, 0.005%, and 0.003% are about half the ATP value as compared with 0.100% and 0.050%, the ATP value at the time of recovery is the same level as or higher than those, and therefore, it is known that the recovery efficiency of cells (single cells) is improved by reducing the amount of the substrate.

Test example 10 cultivation under shaking conditions (comparison with stationary cultivation)

A culture medium composition was prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 (PromoCell Co., #C-28009) so that the final concentration was 0.100% (w/v) or 0.020% (w/v), respectively.

Next, mesenchymal stem cells (CellSource, manufactured by Corning, # 0111201) from human adipose tissue were each suspended in the above-described respective medium compositions so as to be 3X 10 ⁴ cells/mL, and then inoculated into a 100mm flat bottom ultra-low adhesion surface dish (manufactured by Corning, # 3262) at 30 mL/dish. Cells were cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) in a stationary state or in a horizontal shaking state on an in vitro shaking device (manufactured by TAITEC Co., ltd., wave-SI slide, SPEED: 15). The external photographs on day 0 and day 3 of the culture are shown in fig. 13, and the microscopic observation images are shown in fig. 14. From the observation results, the following conditions were confirmed: compared to static culture, shaking culture forms more uniform spheroids.

Culture was continued until day 8 post inoculation, at which point 30mL total was recovered from each dish to a 50mL conical tube. After allowing to stand for 15 minutes, the spheroid composition was allowed to naturally settle, and then the remaining culture supernatant was removed. To this, HBSS (-) (manufactured by Thermo Fisher Co., # 14175095) (20 mL) was added, and the mixture was allowed to stand for 15 minutes again, and after allowing the spheroid composition to naturally settle, the lower 5mL was left, and the supernatant was removed and washed. Next, a liquid (400. Mu.L) in which Liberase (manufactured by Merck Co., # 5401119001) (5 mg) was dissolved in HBSS (-) (2 mL), and TRYPLE SELECT Enzyme (10X) phenol red-free (manufactured by Thermo Fisher Co., # A1217701) (1.4 mL) were added, and the mixture was incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 1 hour in a stationary state, whereby cells were detached from the substrate. The liquid (15 mL) obtained by diluting the obtained cell/substrate suspension (A) with HBSS (-) (8.2 mL) was filtered through a cell sieve (pluriSelect, manufactured by the company, # 43-50070-51) having a mesh size of 70 μm, to thereby obtain a filtrate (B) (15 mL) containing cells separated from the substrate. Further, the filtrate was backwashed with HBSS (-) (15 mL) to obtain a filtrate suspension (C) (15 mL).

The cell concentration of the suspension C was measured by a cell counter (TC-20, manufactured by BIO-RAD Co., ltd.), and the number of the cells recovered was calculated. The number of cells recovered at each concentration and with or without shaking is shown in Table 12. It was confirmed that the cell yield was higher in the transverse oscillation condition than in the resting condition at any concentration. This indicates that not only stirring but also shaking promotes the formation of a spheroid containing a base material, and that subsequent recovery of single cells is also improved by forming the spheroid.

TABLE 12

Test example 11 single cell comparison of spheres

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 25rpm for 11 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. On days 4 and 7 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange. For the comparative example, cells were inoculated in an amount of 4 or 8X 10 ³ cells/well/200. Mu.L into PrimeSurface (registered trademark) plate 96U (manufactured by SUMITOMO BAKELITE Co., #MS-9096U) and subjected to stationary culture in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 4 days. The culture medium used in the comparative example was mesenchymal stem cell proliferation medium 2 to which the base material of preparation example 1 was not added.

(Pretreatment)

The uniformly suspended culture solution was passed through a cell sieve (pluriSelect, manufactured by Corp., # 43-50400-03) having a pore diameter of 400 μm, and then D-PBS (-) (manufactured by Fuji film and Wako pure chemical industries, ltd., # 045-29795) was added to wash the spheres trapped on the sieve, the sieve was turned upside down, and the spheres trapped on the sieve were washed with an appropriate amount of D-PBS (-), whereby 0.9mL of the cell suspension was recovered and transferred to a 12-well plate (manufactured by Corning Co., # 351143). This condition was defined as example 3. In the comparative example, spheres were collected from the plate into a 15mL tube, allowed to settle naturally, the supernatant was removed, D-PBS (-) was added, allowed to settle naturally again, and the supernatant was removed, whereby the spheres were washed, suspended in 0.9mL of D-PBS (-), and transferred to a 12-well plate. The condition using the spheres obtained by seeding 4×10 ³ cells/well was used as comparative example 2, and the condition using the spheres obtained by seeding 8×10 ³ cells/well was used as comparative example 3.

(Enzyme treatment, cell staining)

10. Mu.L of a solution of Calcein-AM (manufactured by Tokugaku Co., #C326) dissolved in DMSO at a final concentration of 0.5mg/mL was added to 0.5mL of D-PBS (-) or TrypLE (registered trademark) Select Enzyme (10X) phenol red-free (manufactured by Thermo Fisher Co., #A 1217701), 0.1mL was added to a 12-well plate, and the plate was incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 30 minutes. Then, 20 shots were performed using Eppendorf Research (registered trademark) plus100 to 1000 μl (manufactured by Eppendorf corporation, # 3120000062) with a discharge amount of 0.5mL under the condition that TrypLE was added.

(Image acquisition)

After pretreatment and enzyme treatment, the whole wells of the 12-well plate were photographed using Cell3iMagerduos (manufactured by sceen holders co., ltd.) to obtain a bright field image and a living Cell-specific fluorescent staining image. The image is shown in fig. 15. Using the acquired images, the average diameter of the spheres was calculated using the Cell3iMagerduos built-in software. The results are shown in Table 13. Spheres with unclear contours, spheres with overlapping spheres, spheres with contours that cannot be accurately identified are excluded from the analysis object. An image of the sphere used in the analysis is shown in fig. 16 in green.

TABLE 13

As shown in table 13, the spheres of example 3 had a larger average diameter than those of comparative examples 2 and 3 after pretreatment, but as shown in fig. 15, the contours of the spheres of example 3 disappeared and dispersed into single cells by the enzyme treatment and blowing. On the other hand, the spheres of comparative examples 2 and 3 were not completely dispersed into single cells. In addition, the dispersed single cells in example 3 were stained with fluorescence, thus indicating living cells. From the above results, it was revealed that the spheres formed using the substrate 1 can have higher dispersibility into single cells than spheres formed without the substrate.

Test example 12 dispersion of spheres Using cell dispersing tool

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 15cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 25mL of a culture medium composition prepared by adding the substrate of preparation example 1 to the mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 25rpm for 10 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. On days 4 and 7 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange.

(Separation of spheres)

On day 10 of the culture, the whole amount of the culture solution was passed through a cell sieve (pluriSelect, inc. # 43-50200-03) having a pore size of 200 μm, and then washed with 30mL of D-PBS (-), the net was turned upside down, and the spheres trapped on the net were recovered with 10mL of D-PBS (-).

(Enzyme treatment)

Mu.L of Liberase (registered trademark) TM RESEARCH GRADE (manufactured by Merck Co., # 5401119001) solution and 2mL of TrypLE (registered trademark) Select Enzyme (10X) having no phenol red (manufactured by Thermo Fisher Co., # A1217701) were mixed with 17446. Mu.L of D-PBS (-) at a final concentration of 13U/mL, to prepare 20mL of an Enzyme solution. After the separated spheres were subjected to centrifugal separation (400×g, 3 min, deccel mode), the supernatant was removed, suspended in an enzyme solution heated to 37 ℃, and transferred to a cell dispersion tool (made by ABLE Corporation). The cell dispersion tool was set in a high-rotation stirrer (made by ABLE Corporation) with a temperature adjusting function for the dispersion tool, which had been heated to 37℃to disperse the cells at 1200 rpm.

(Microscopic observation)

At the time points of enzyme treatments of 0, 3, 5, 10, 15, 20, and 30 minutes, 0.5mL of the uniformly suspended suspension was transferred to a 12-well plate (manufactured by Corning corporation, # 351143) and observed using an inverted microscope (manufactured by Olympus Corporation, # IX 73). The scale bar represents 500. Mu.m.

As shown in fig. 17, the spheres disintegrate with time, and single cells are released. From the above results, it was demonstrated that spheres formed using the substrate can be separated into single cells.

Test example 13 dispersion of spheres in reactor

A medium containing mesenchymal stem cell proliferation medium 2 (manufactured by PromoCell Co., # C-28009) and penicillin-streptomycin solution (x 100) (manufactured by Fuji photo-Paul et al, # 168-23191) was added to UniVessel (registered trademark) Glass1L (manufactured by Sartorius Co., ltd.) sterilized (121 ℃ C., 20 minutes) by an autoclave, and the mixture was connected to BIOSTAT (registered trademark) B-DCU (manufactured by Sartorius Co., ltd.) to adjust the medium for 30 minutes under conditions of compressed air of 130ccm and CO ₂ of 6ccm, 37 ℃ C., 60 rpm. For mesenchymal stem cells derived from human umbilical cord (PromoCell, inc. # C-12971), mesenchymal stem cell proliferation medium 2 was used, and the culture was performed on a 15cm dish (Corning, inc. # 430167) for 3 days. Then, cells were removed by using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the resulting mixture was added to a medium in which the final concentration of the substrate of preparation example 1 was adjusted so as to be 0.05% (w/v) in advance, at an inoculation concentration of 3X 10 ⁴ cells/mL. The total amount of medium was 450mL. For the cultivation, the agitation cultivation was performed under conditions of 130ccm of compressed air and 6ccm of CO ₂ at 37℃and 60rpm for 10 days. On days 4 and 7 of the culture, half of the cell suspension of the reactor was recovered and centrifuged (300×g, 3 min, deccel mode), and then the supernatant was removed, suspended in a new medium, and returned to the reactor, whereby the medium was replaced.

(Separation of spheres)

On day 10 of the culture, 50mL of the culture solution was passed through a cell sieve (pluriSelect, inc. # 43-50200-03) having a pore diameter of 200 μm, and then washed with 40mL of HBSS (-) (Thermo Fisher, inc. # 14175095), and the net was turned upside down, and HBSS (-) was added, whereby the spheres trapped on the net were recovered. This operation was repeated, and the spheres were collected in an amount of 340mL of the culture medium.

(Enzyme treatment)

A50 mL Enzyme solution was prepared by mixing 1385. Mu.L of Liberase (registered trademark) TM RESEARCH GRADE (manufactured by Merck Co., # 5401119001) solution, 5mL of TrypLE (registered trademark) Select Enzyme (10X) free of phenol red (manufactured by Thermo Fisher Co., # A1217701), and 43615. Mu.L of HBSS (-) in a final concentration of 13U/mL. After the separated spheres were subjected to centrifugal separation (400×g, 3 min, deccel mode), the supernatant was removed, suspended in an enzyme solution heated to 37℃and transferred to a 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A), and the mixture was placed on a 6-gang stirrer (6-position program stirrer) (manufactured by WakenBtech Co., # WKN-1106-P) and treated at 150rpm in a CO ₂ incubator (37℃5% CO ₂) for 25 minutes, whereby the cells were dispersed. The dispersed suspension was passed through a cell sieve (manufactured by Nissan chemical Co., ltd.) having a pore size of 65 μm, and the substrate was removed to obtain a filtrate.

(Microscopic observation)

0.5ML of the culture medium at each stage of the uniform suspension was transferred to a 12-well plate (manufactured by Corning Co., # 351143), and observed using an inverted microscope (manufactured by Olympus Corporation, # IX 73). The cells and the substrate were stained specifically with trypan blue solution (Fuji photo-pure Co., # 207-17081) before and after the cell screen treatment, and then added to a counting plate (Bio-Rad Co., # 1450011), followed by the same observation. The acquired image is shown in fig. 18. The scale bar represents 500. Mu.m.

As shown in fig. 18, the fine substrate was removed by sphere separation, and the spheres were disintegrated and single cells were dissociated by enzyme treatment using a bioreactor. Further, the substrate is removed by treating the cell screen. From the above results, it was revealed that spheres formed using the substrate can be dispersed into single cells in the bioreactor, and the substrate can be removed by using a cell screen.

Test example 14 Gene expression analysis of mesenchymal Stem cells cultured under suspension and stirring

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, the cells were peeled off using DETACHKIT (manufactured by PromoCell Co., #C-41210). The cells thus obtained were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to the mesenchymal stem cell growth medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and the mixture was stirred and cultured in a CO ₂ incubator (37 ℃, 5% CO ₂). As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and stirring was performed using a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) with no break at 25 rpm. On day 4 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange. As a comparison object, cells were inoculated into a 6-well adhesion culture plate (# 3516, manufactured by Corning Co., ltd.) so as to be 8X 10 ⁴ cells/well/2 mL, and adhesion culture was performed. On day 4 of culture, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), inoculated so as to be 1X 10 ⁵ cells/well/2 mL, and further cultured by adhesion for 3 days.

(Analysis of Gene expression)

Cells were collected on day 0 and day 7 of the culture, and 300. Mu.L of RLT solution (RNEASY MINI KIT (manufactured by QIAGEN Co., # 74106)) was added to prepare an RNA extraction solution. After 300. Mu.L of 70% ethanol was added to the RNA extraction solution, the solution was applied to an RNeasy centrifuge column and centrifuged at 8000 Xg for 15 seconds. Next, 700. Mu.L of RW1 solution was added to the RNeasy column and centrifuged at 8000 Xg for 15 seconds. Next, 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 15 seconds. Further 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 2 minutes. RNase-free solution was added to RNA present in the RNeasy centrifugation column and eluted. Next, cDNA was synthesized from the obtained RNA using PRIMESCRIPT RT REAGENT KIT (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 037A). Real-time PCR was performed using the synthesized cDNA and Premix EX Taq (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 039A) and Taq man probe (manufactured by Applied Bio Systems). As Taq man probe (Applied Bio Systems Co.), TSPAN7 uses Hs00190284 _m1, MFAP4 uses Hs00412974 _m1, CD55 uses Hs00892618 _m1, GPX3 uses Hs00173566 _m1, HMOX1 uses Hs01110250 _m1, RAB27B uses Hs00188156 _m1, IL33 uses Hs00369211 _m1, GAPDH uses Hs99999905 _m1. The device uses REAL TIME PCR to 7500. For analysis, relative values obtained by correcting the values of the target genes with the values of GAPDH were calculated, and the cells on day 0 were compared with 1. The results are shown in Table 14.

TABLE 14

As shown in table 14, it was found that expression of CD55, HMOX1, TSPAN7, RAB27B, IL, GPX3, and MFAP4 was increased in the mesenchymal stem cells subjected to suspension culture with stirring, as compared with the mesenchymal stem cells subjected to adhesion culture.

Test example 15 Effect of stirred culture on extracellular vesicle production

Mesenchymal stem cells derived from human umbilical cord (manufactured by PromoCell Co., # C-12971) and mesenchymal stem cells derived from human fat (manufactured by CellSource Co., # 0111201) were subjected to 3-day adherent culture on a 10cm dish (manufactured by Corning Co., # 430167) using mesenchymal stem cell proliferation medium 2 (manufactured by PromoCell Co., # C-28009). Then, the cells were peeled off using DETACHKIT (manufactured by PromoCell Co., #C-41210). The cells obtained were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to the mesenchymal stem cell growth medium 2 at a final concentration of 0.05% (w/v) at an inoculation concentration of 3X 10 ⁴ cells/mL, and cultured under stirring in a CO ₂ incubator (37 ℃, 5% CO ₂). As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and stirring was performed using a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) with continuous stirring (stirring culture set) at 25rpm (mesenchymal stem cells derived from human umbilical cord) and 40rpm (mesenchymal stem cells derived from human fat). On day 3 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange, followed by further culture until day 7. The culture broth was transferred to a 50mL centrifuge tube on day 7 of incubation, and then centrifuged at 300 Xg for 3 minutes to remove the medium. next, 30mL of D-PBS was added to the cells, which were centrifuged at 300 Xg for 3 minutes, and the D-PBS was removed. After the same procedure was performed again, 20mL of D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) (Fuji film and Wako pure chemical industries, ltd. # 043-30085) containing 10% exosome-free fetal bovine serum (manufactured by Gibco Co., ltd. # 174952) was added, and inoculated into T75 Nunclon SPHERA EASYFLASK (manufactured by Thermo Fisher Co., ltd. # 174952) in a CO ₂ incubator (37 ℃ C., pH, 5% CO ₂) for 2 days. After 2 days, the culture supernatant was recovered. At the time of cell inoculation, 250. Mu.L of the cell suspension was collected on days 7 and 9 of culture, an equal amount of CellTiter-Glo (registered trademark) luminescence cell viability assay (manufactured by Promega Co.) was added, and the luminosity was measured by using Enspire (manufactured by Perkinelmer Co.) to determine the number of cells at each time point. As a comparison object, cells were inoculated into 100mm dishes (manufactured by Corining Co., # 430167) so as to be 9X 10 ⁵ cells/10 mL/dish, and subjected to adhesion culture (adhesion culture group). After removal of the medium on day 2 of the culture, 30mL of D-PBS was added and removed, and the above procedure was repeated 2 times. Next, 10mL of D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) containing 10% exosome-free fetal bovine serum was added, and the mixture was cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 2 days. after 2 days, the culture supernatant was recovered, and the cells were recovered using DETACHKIT, and the cell number was determined.

(Recovery of extracellular vesicles by ultracentrifugation and particle count measurement)

The collected culture supernatant was centrifuged at 2000 Xg for 10 minutes, and the supernatant was collected and passed through a 0.22 μm filter (Millipore Co., # SLGSR SB). The treated culture supernatant was added to a UC tube (manufactured by Beckman Coulter, # 344059), placed on SW41Ti (manufactured by Beckman Coulter) and centrifuged at 35000rpm and 4℃for 70 minutes using Optima L-90K. After centrifugation, the supernatant was removed, 10mL of D-PBS was added to the UC tube, and the mixture was centrifuged at 35000rpm at 4℃for 70 minutes. After centrifugation, the supernatant was removed and suspended in 100. Mu.L of D-PBS. The recovered extracellular vesicles were subjected to Particle count measurement using ZetaView (manufactured by Particle Metrix Co.). The number of extracellular vesicles produced per unit cell was calculated by dividing the number of particles obtained by the number of cells at the time point when the culture supernatant was obtained. The results are shown in Table 15.

TABLE 15

As shown in table 15, in any of umbilical cord-derived and adipose-derived mesenchymal stem cells, the amount of extracellular vesicles obtained by agitation culture and the amount of extracellular vesicles per unit cell were larger than those obtained by adhesion culture.

(ELISA-based measurement of exosome marker CD 63)

For detection of CD63, PS Capture (trademark) exosome ELISA kit (anti-mouse IgG POD) (manufactured by Fuji film and Wako pure chemical industries, ltd. # 297-79201) was used. The Reaction/Washing solution (1×) was prepared by adding Exosome Binding Enhancer (100×) in an amount of one percent to the Reaction/Washing Buffer (1×) prepared by diluting the Reaction/Washing Buffer (10×) 10 times with purified water. Each culture broth was diluted 500-fold with the reaction/wash solution (1×). After 3 washes of the Exosome Capture 96-well plate with 300 μl of reaction/wash solution (1×), 500-fold diluted extracellular vesicles were added to each well and allowed to shake with a microplate shaker while reacting at room temperature for 2 hours. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and 100. Mu.L of Control Primary Antibody Anti-CD63 (. Times.100) diluted 1000-fold with the reaction/washing solution (1X) was added thereto, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of Secondary Antibody HRP-conjugated Anti-mouse IgG (100X) diluted 1000-fold with the reaction/washing solution (1X) was added, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 5 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of TMB solution was added thereto, and after shaking for 1 minute with a microplate shaker, the reaction was performed at room temperature for 30 minutes. After completion of the reaction, 100. Mu.L of a stop solution was added, and the mixture was shaken with a microplate shaker for 5 seconds, and then absorbance at 450nm was measured with Enspire (manufactured by Perkinelmer). The results are shown in Table 16.

TABLE 16

As shown in Table 16, expression of CD63 was observed under any conditions, and a stronger signal was obtained in the agitation culture than in the adhesion culture. From the above, it was revealed that the production of exosomes of mesenchymal stem cells can be promoted by suspension culture with stirring.

Test example 16 Effect of stirred culture on extracellular vesicle production 2

For mesenchymal stem cells derived from human fat (CellSource, manufactured by # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, manufactured by # C-28009) was used, and the cells were subjected to adhesion culture on a 10cm dish (manufactured by Corning, manufactured by # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and suspended in 30mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 at a final concentration of 0.05% (w/v) in a CO ₂ incubator (37 ℃ C., 3 ℃ C., 3: ⁴ cells/mL), 5% CO ₂) were cultured with stirring. As a culture vessel, a30 mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used for stirring, and the stirring was performed at 50rpm without interruption (stirring culture set). In addition, as a comparative example of the agitation culture, cells were suspended in 30mL of mesenchymal stem cell proliferation medium 2 containing 600mg of Corning (registered trademark) Low Concentration Synthemax (registered trademark) II Microcarriers (manufactured by Corning Corp., # 3781) at an inoculation concentration of 3X 10 ⁴ cells/mL, and cultured in a CO ₂ incubator (37 ℃ C.,), 5% CO ₂) were cultured with stirring. as a culture vessel, a 30mL disposable reactor was used, and stirring was performed using a special magnetic stirrer, and after repeating the operation of standing for 59 minutes and stirring at 55rpm for 1 minute 10 times, continuous stirring was performed at 55rpm (microcarrier culture group). On day 4 of incubation, the above culture broth was transferred to a 50mL centrifuge tube, and then centrifuged at 300 Xg for 3 minutes to remove the medium. Next, 30mL of D-PBS was added, and after centrifugation at 300 Xg for 3 minutes, the D-PBS was removed. After the same procedure was performed again, 30mL of D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) (manufactured by Fuji film and Wako pure chemical industries, ltd. # 043-30085) containing 10% exosome-free fetal bovine serum (manufactured by Gibco Co., ltd., A2720801) was added, and the mixture was inoculated again into a special magnetic stirrer, and stirred continuously at 50rpm or 55rpm for 2 days. After 2 days, the culture supernatant was recovered. At the time of cell inoculation and on day 6, 250. Mu.L of the cell suspension was separated, an equal amount of CellTiter-Glo (registered trademark) was added to the cell viability assay (manufactured by Promega corporation), and the luminosity was measured by using Enspire (manufactured by Perkinelmer corporation), thereby measuring the number of cells at each time point. As the adherent culture, cells were inoculated into 100mm dishes (manufactured by Corining Co., # 430167) so as to be 9X 10 ⁵ cells/10 mL/dish, and adherent culture (adherent culture group) was performed. the medium was removed on day 2 of culture, then 30mL of D-PBS was added and removed, and the above procedure was repeated 2 times. Next, 10mL of D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) containing 10% exosome-free fetal bovine serum was added, and the mixture was cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 2 days. after 2 days, the culture supernatant was recovered, and the cells were recovered using DETACHKIT, and the cell number was determined. On days 0, 4 (adhesion culture) and 6 (stirring culture and microcarrier culture), 300. Mu.L of RLT solution (RNEASY MINI KIT (manufactured by QIAGEN Co., # 74106) was added to prepare an RNA extraction solution, and on days 0, 4 (adhesion culture) and 6 (stirring culture and microcarrier culture), 150. Mu.L of RIPA buffer (manufactured by Fuji film and Wako pure chemical industries, ltd. # 182-02451) containing a single-use mixture (100X) of 1 XHalt (trademark) protease and phosphatase inhibitor was used to prepare a whole cell lysate.

The collected culture supernatant was centrifuged at 2000 Xg for 10 minutes, and the supernatant was collected and passed through a 0.22 μm filter (Millipore Co., # SLGSR SB). The treated culture supernatant was added to a UC tube (manufactured by Beckman Coulter, # 344059), placed on SW41Ti (manufactured by Beckman Coulter) and centrifuged at 35000rpm and 4℃for 70 minutes using Optima L-90K. After centrifugation, the supernatant was removed, 10mL of D-PBS was added to the UC tube, and the mixture was centrifuged at 35000rpm at 4℃for 70 minutes. After centrifugation, the supernatant was removed, and suspended in 50. Mu.L of D-PBS for the adherent culture group, and in 100. Mu.L of D-PBS for the microcarrier culture and the stirred culture group. The recovered extracellular vesicles were subjected to Particle count measurement using ZetaView (manufactured by Particle Metrix Co.). The number of extracellular vesicles produced per unit cell was calculated by dividing the number of particles obtained by the number of cells at the time point when the culture supernatant was obtained. The results are shown in Table 17.

TABLE 17

Culture conditions	10 ⁹ Particles/ml	10 ⁹ Particles/ml/10 ⁶ cells
			Adhesion culture	2.2	0.1
Microcarrier culture	15	0.8
			Stirring culture	33	5.3

As shown in Table 17, the number of extracellular vesicles per unit cell was increased in the agitation culture as compared with the adhesion culture and the microcarrier culture.

(ELISA-based determination of extracellular vesicle markers)

For detection of CD63, PS Capture (trademark) exosome ELISA kit (anti-mouse IgG POD) (manufactured by Fuji film and Wako pure chemical industries, ltd. # 297-79201) was used. The Reaction/Washing solution (1×) was prepared by adding Exosome Binding Enhancer (100×) in an amount of one percent to the Reaction/Washing Buffer (1×) prepared by diluting the Reaction/Washing Buffer (10×) 10 times with purified water. Considering the amount of medium used and the amount of solution suspended after ultracentrifugation, the adherent culture group was performed using a 400-fold diluted extracellular vesicle solution, and the microcarrier culture and stirred culture group was performed using a 600-fold diluted reaction/wash solution (1×). After washing ExosomeCapture 96-well plates 3 times with 300. Mu.L of the reaction/washing solution (1X), 100. Mu.L of the diluted extracellular vesicle solution was added to each well, and the mixture was shaken with a microplate shaker while reacting at room temperature for 2 hours. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and 100. Mu.L of Control Primary Antibody Anti-CD63 (. Times.100) diluted 1000-fold with the reaction/washing solution (1X) was added thereto, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of Secondary Antibody HRP-conjugated Anti-mouse IgG (100X) diluted 1000-fold with the reaction/washing solution (1X) was added, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 5 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of TMB solution was added thereto, and after shaking for 1 minute with a microplate shaker, the reaction was performed at room temperature for 30 minutes. After completion of the reaction, 100. Mu.L of a stop solution was added, and the mixture was shaken with a microplate shaker for 5 seconds, and thereafter, the absorbance at 450nm was measured with Enspire (manufactured by Perkinelmer). The absorbance of the background was subtracted from each group to obtain Δabs. The results are shown in Table 18.

TABLE 18

	ΔAbs
		Adhesion culture	0.314
Microcarrier culture	0.534
		Stirring culture	0.619

As shown in table 18, expression of CD63 was observed under any conditions, but the strongest signal was confirmed in the stirred culture.

(Confirmation of RAB27B expression level based on Gene expression analysis)

After 300. Mu.L of 70% ethanol was added to the RNA extraction solution, the solution was applied to an RNeasy centrifuge column and centrifuged at 8000 Xg for 15 seconds. Next, 700. Mu.L of RW1 solution was added to the RNeasy column and centrifuged at 8000 Xg for 15 seconds. Next, 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 15 seconds. Further 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 2 minutes. RNase-free solution was added to RNA present in the RNeasy centrifugation column and eluted. Next, cDNA was synthesized from the obtained RNA using PRIMESCRIPT RT REAGENT KIT (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 037A). Real-time PCR was performed using the synthesized cDNA and Premix EX Taq (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 039A) and Taq man probe (manufactured by Applied Bio Systems). As Taq man probe (Applied Bio Systems Co.), hs00188156-m1 was used for RAB27B, and Hs99999905-m1 was used for GAPDH. The apparatus used was QuantStudio (manufactured by Thermo Fisher Co.). For analysis, relative values obtained by correcting the values of the target genes with the values of GAPDH are calculated and compared.

TABLE 19

	Relative value of
		Day 0	0.03
Adhesion culture	0.26
		Microcarrier culture	0.61
Stirring culture	1.16

As shown in Table 19, an increase in the mRNA expression level of RAB27B was observed under agitation culture.

(Confirmation of expression variation of RAB27B protein)

The electrophoresis tank was filled with Ezrun C + solution (ATTO, # 2332320) as a buffer. As a gel for electrophoresis, E-T12.5L E-PAGEL 12.5.5% (manufactured by ATTO Co., # 2331820) was set, and each sample was loaded in 12. Mu.g/lane. Electrophoresis was performed at 100V for 70 minutes. After electrophoresis, transfer was performed to the film for 7 minutes under conditions of 1.3A and 25V using Trans-Blot Turbo MINI PVDF TRANSFER PACK (manufactured by Bio-Rad Co., # 1704156). After transfer, the film was immersed in a TBS-T solution prepared using Tris Buffered SALINE WITH TWEEN (registered trademark) 20 (TBS-T) tablet, pH7.6 (manufactured by Takara Bio Inc.. Co., #T9142) and shaken at room temperature for 1 hour. Then, the mixture was immersed in PVDF sealer CAN GET SIGNAL (registered trademark) (TOYOBO, # NYPBR 01) and shaken at room temperature for 3 hours. The membrane was immersed in a TBS-T Solution, shaken 1 time at 15 minutes and 2 times at 5 minutes, then immersed in Anti RAB27B diluted 2000-fold with CAN GET SIGNAL Solution 1 (manufactured by TOYOBO Co., # NKB-201), human (Rabbit) Unlabeled (manufactured by Peprotech Co., # 13412-1-AP) and β -actin (D6A 8) mAb diluted 2000-fold (manufactured by CELL SIGNALING TECHNOLOGY Co., # 8457), and shaken overnight at 4 ℃. The next day, the membrane was immersed in TBS-T Solution and shaken 3 times for 20 minutes, and then immersed in Anti-Rabbit IgG diluted 5000-fold with CAN GET SIGNAL Solution 2 (manufactured by TOYOBO Co., # NKB-301) and HRP-Linked Whole Ab Donkey (manufactured by Cytiva Co., # NA934-1 ML) and shaken at room temperature for 1 hour. The film was immersed in a TBS-T solution, and then shaken 1 time for 15 minutes and 1 hour, followed by light emission using ImmunoStar (registered trademark) Zeta (manufactured by Fuji film and Wako pure chemical industries, ltd., # 297-72403). The detection was performed using ChemiDoc XRS Plus (manufactured by Bio-Rad Co.). The results are shown in FIG. 19.

As shown in FIG. 19, an increase in the expression level of RAB27B protein was observed under agitation culture.

Test example 17 Signal pathway analysis of mesenchymal Stem cells cultured by the method of the present invention

For mesenchymal stem cells derived from human fat (CellSource, manufactured by # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, manufactured by # C-28009) was used, and the cells were subjected to adhesion culture on a 10cm dish (manufactured by Corning, manufactured by # 430167) for 3 days. Then, the cells were peeled off using DETACHKIT (manufactured by PromoCell Co., #C-41210). The detached cells were suspended in mesenchymal stem cell proliferation medium 2 (30 mL) containing the substrate of preparation example 1 (final concentration 0.05% (w/v)) at an inoculation concentration of 3X 10 ⁴ cells/mL, and cultured under agitation in a CO ₂ incubator (37 ℃, 5% CO ₂). As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used for stirring, and the stirring was performed at 50rpm without interruption (stirring culture set). Culture the culture vessel was allowed to stand for 10 minutes on day 3, and half of the culture supernatant was subjected to medium exchange, and culture was continued until day 7. As a control, an adhesion culture (adhesion culture group) was performed on a 10cm dish (manufactured by Corning Co. # 430167) for 3 days. Nuclear fraction (Nuclear fractions) was obtained from cells cultured by adhesion and agitation on days 3 and 7 of the culture using Nuclear Extraction Kit (manufactured by Raybio Co., #NE-50).

(Confirmation of expression of NFE2L2 (also referred to as "NRF 2"), P65 and phosphorylated P65 (P-P65) proteins by Western blotting)

The electrophoresis tank was filled with EzrunC + solution (ATTO, # 2332320) as a buffer. As a gel for electrophoresis, E-T12.5L ePAGEL 12.5.5% (manufactured by ATTO Co., # 2331820) was set, and each sample was loaded in 12. Mu.g/lane. Electrophoresis was performed at 100V for 70 minutes. Transfer was performed on the film for 7 minutes under conditions of 1.3A and 25V using Trans-Blot Turbo MINI PVDF TRANSFER PACK (manufactured by Bio-Rad Co., # 1704156). After transfer, the film was immersed in a TBS-T solution prepared using TrisBuffered SALINE WITH TWEEN (registered trademark) 20 (TBS-T) tablets, pH7.6 (manufactured by Takara Bio Inc. # T9142), and shaken at room temperature for 1 hour. Then, the mixture was immersed in PVDF sealer CAN GET SIGNAL (registered trademark) (TOYOBO, # NYPBR 01) and shaken at room temperature for 3 hours. The membrane was immersed in a TBS-T Solution, and then immersed in a Solution of CAN GET SIGNAL solutions 1 (TOYOBO, # NKB-201) diluted 1000-fold with a XPR Rabbit mAb (CELL SIGNALING TECHNOLOGY, # 12721) diluted 1000-fold with a Solution of CAN GET SIGNAL solutions 1 (TOYOBO, # NKB-201); RELA, human (Rabbit) Unlabeled (Peprotech, # 10745-1-AP) diluted 1000-fold with Phospho-NF-kB p65 (Ser 536) (93H 1) Rabbit mAb (CELL SIGNALING Technology, # 3033) was shaken overnight at 4 ℃. The membrane was immersed in TBS-T Solution, shaken 3 times for 20 minutes, and then immersed in Anti-Rabbit IgG diluted 5000-fold with CAN GET SIGNAL Solution 2 (manufactured by TOYOBO Co., # NKB-301), HRP-Linked Whole Ab Donkey (manufactured by Cytiva Co., # NA934-1 ML), and shaken at room temperature for 1 hour. The film was immersed in a TBS-T solution, and then shaken 1 time for 15 minutes and 1 hour, followed by light emission using ImmunoStar (registered trademark) Zeta (manufactured by Fuji film and Wako pure chemical industries, ltd., # 297-72403). The detection was performed using ChemiDoc XRS Plus (manufactured by Bio-Rad Co.). The results are shown in FIG. 20.

As shown in FIG. 20, it was revealed that NFE2L2 in the nucleus and P65 and P-P65, which are subunits of NF-kB, were increased by the agitation culture as compared with the adhesion culture.

Test example 18 analysis of mechanism of increase in production of extracellular vesicles from mesenchymal stem cells cultured with stirring

For mesenchymal stem cells derived from human fat (CellSource, manufactured by # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, manufactured by # C-28009) was used, and the cells were subjected to adhesion culture on a 10cm dish (manufactured by Corning, manufactured by # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell, # C-41210) and inoculated into a 6-well cell culture plate (manufactured by Corning, # 3516) so that the inoculation concentration became 5X 10 ⁵ cells/1.75 mL. Meanwhile, a sample containing 7.5. Mu.L Lipofectamine RNAiMAX Transfection Reagent (manufactured by Thermo Fisher Co., # 13778075), 25pmol of SILENCER SELECT NEGATIVE control#1siRNA (hereinafter sometimes referred to as "Neg") (manufactured by Thermo Fisher Co., # 4390843), RAB27B (# s 11696), NFE2L2 (# s 9493), Opti-MEM (trademark) I Reduced Serum Medium (manufactured by Thermo Fisher Co., ltd., # 31985070) of TLR2 (# s 169) (manufactured by Thermo Fisher Co., ltd.) was added to each well at 250. Mu.L/well. After 1 day, the medium was removed, cells were detached using DETACHKIT, and suspended in 5mL or 30mL of a mesenchymal stem cell proliferation medium 2 medium composition containing the substrate of preparation example 1 at a final concentration of 0.05% (w/v) so as to be 3X 10 ⁴ cells/mL, in a CO ₂ incubator (37 ℃ C., 3 ℃ C., 3X 24 cells/mL, 5% CO ₂) were cultured with stirring. As the culture vessel, a 5mL disposable reactor (manufactured by ABLE Corporation, # ABBWVS A) or a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # ABBWBP N0S-6 or #BWS-S03N 0S-6) was used for stirring, and the stirring was performed at 50rpm without interruption (stirring culture group). After 2 days, the whole medium containing cells and substrate was transferred to a centrifuge tube and centrifuged at 300 Xg for 3 minutes. The centrifuged culture supernatant was recovered for ELISA assay. Further, on days 0 and 3, cells were collected, and 300. Mu.L of RLT solution (RNEASY MINI KIT (manufactured by QIAGEN, # 74106) was added to prepare an RNA extraction solution, and on days 0 and 3, a single use (100X) of RIPA buffer (manufactured by Fuji film and Wako pure chemical industries, ltd. # 182-02451) containing 150. Mu.L of a mixture of 1 XHalt (trademark) protease and phosphatase inhibitor was used to prepare a whole cell lysate.

(Confirmation of RAB27B expression level based on Gene expression analysis)

After 300. Mu.L of 70% ethanol was added to the RNA extraction solution, the solution was applied to an RNeasy centrifuge column and centrifuged at 8000 Xg for 15 seconds. Next, 700. Mu.L of RW1 solution was added to the RNeasy column and centrifuged at 8000 Xg for 15 seconds. Next, 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 15 seconds. Further 500. Mu.L of RPE solution was added and centrifuged at 8000 Xg for 2 minutes. RNase-free solution was added to RNA present in the RNeasy centrifugation column and eluted. Next, cDNA was synthesized from the obtained RNA using PRIMESCRIPT RT REAGENT KIT (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 037A). Real-time PCR was performed using the synthesized cDNA and Premix EX Taq (PERFECT REAL TIME) (manufactured by Takara Bio Inc. # RR 039A) and Taq man probe (manufactured by Applied Bio Systems). As Taq man probe (Applied Bio Systems Co.), hs00188156 _m1 was used for RAB27B, hs99999905 _m1 was used for GAPDH. The apparatus used was QuantStudio (manufactured by Thermo Fisher Co.). For analysis, relative values obtained by correcting the values of the target genes with the values of GAPDH are calculated and compared. The results are shown in Table 20.

TABLE 20

As shown in table 20, it was confirmed that the mRNA expression amount of RAB27B was reduced by siRNA of RAB27B in the agitation culture. In addition, a decrease in the mRNA expression level of RAB27B was also observed at the time of siRNA treatment of NFE2L2 or TLR 2.

(Confirmation of RAB27B protein expression variation)

The electrophoresis tank was filled with EzrunC + solution (ATTO, # 2332320) as a buffer. As a gel for electrophoresis, E-T12.5L ePAGEL 12.5.5% (manufactured by ATTO Co., # 2331820) was set, and each sample was loaded in 12. Mu.g/lane. Electrophoresis was performed at 100V for 70 minutes. After electrophoresis, transfer was performed to the film for 7 minutes under conditions of 1.3A and 25V using Trans-Blot Turbo MINI PVDF TRANSFER PACK (manufactured by Bio-Rad Co., # 1704156). After transfer, the film was immersed in a TBS-T solution prepared using TrisBuffered SALINE WITH TWEEN (registered trademark) 20 (TBS-T) tablets, pH7.6 (manufactured by Takara Bio Inc. # T9142), and shaken at room temperature for 1 hour. Then, the mixture was immersed in PVDF sealer CAN GET SIGNAL (registered trademark) (TOYOBO, # NYPBR 01) and shaken at room temperature for 3 hours. The membrane was immersed in a TBS-T Solution, shaken 1 time for 15 minutes and 2 times for 5 minutes, then immersed in an Anti RAB27B diluted 2000-fold with CAN GET SIGNAL Solution 1 (manufactured by TOYOBO Co., # NKB-201), human (Rabbit) Unlabeled (manufactured by Peprotech Co., # 13412-1-AP) and a β -actin (D6A 8) Rabbit mAb diluted 2000-fold (manufactured by CELL SIGNALING TECHNOLOGY Co., # 8457), and shaken overnight at 4 ℃. The next day, the membrane was immersed in TBS-T Solution and shaken 3 times for 20 minutes, and then immersed in Anti-Rabbit IgG diluted 5000-fold with CAN GET SIGNAL Solution 2 (manufactured by TOYOBO Co., # NKB-301) and HRP-Linked Whole Ab Donkey (manufactured by Cytiva Co., # NA934-1 ML) and shaken at room temperature for 1 hour. The film was immersed in a TBS-T solution, and then shaken 1 time for 15 minutes and 1 hour, followed by light emission using ImmunoStar (registered trademark) Zeta (manufactured by Fuji film and Wako pure chemical industries, ltd., # 297-72403). The detection was performed using ChemiDoc XRS Plus (manufactured by Bio-Rad Co.). The results are shown in FIG. 21.

As shown in fig. 21, a decrease in the protein expression level of RAB27B due to siRNA treatment of RAB27B was confirmed in the agitation culture. In addition, a decrease in the protein expression level of RAB27B was also observed at the time of siRNA treatment of NFE2L2 or TLR 2.

Since the mRNA and protein expression level of RAB27B is decreased at the time of siRNA treatment of NFE2L2 or TLR2, when mesenchymal stem cells are cultured on the present substrate, the expression level of RAB27B is increased through TLR2 and NFE2L2, and as a result, the amount of sEV generated is increased.

(ELISA-based determination of extracellular vesicle markers)

For detection of CD63, PS Capture (trademark) exosome ELISA kit (anti-mouse IgG POD) (manufactured by Fuji film and Wako pure chemical industries, ltd. # 297-79201) was used. The Reaction/Washing solution (1×) was prepared by adding Exosome Binding Enhancer (100×) in an amount of one percent to the Reaction/Washing Buffer (1×) prepared by diluting the Reaction/Washing Buffer (10×) 10 times with purified water. After washing ExosomeCapture 96-well plates 3 times with 300. Mu.L of the reaction/washing solution (1X), 100. Mu.L of the obtained culture supernatant was added to each well, and the mixture was shaken with a microplate shaker while being reacted at room temperature for 2 hours. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and 100. Mu.L of Control Primary Antibody Anti-CD63 (. Times.100) diluted 1000-fold with the reaction/washing solution (1X) was added thereto, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 3 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of Secondary Antibody HRP-conjugated Anti-mouse IgG (100X) diluted 1000-fold with the reaction/washing solution (1X) was added, and the mixture was shaken with a microplate shaker and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was discarded, each well was washed 5 times with 300. Mu.L of the reaction/washing solution (1X), and then 100. Mu.L of TMB solution was added thereto, and after shaking for 1 minute with a microplate shaker, the reaction was performed at room temperature for 30 minutes. After completion of the reaction, 100. Mu.L of a stop solution was added, and the mixture was shaken with a microplate shaker for 5 seconds, and thereafter, the absorbance at 450nm was measured with Enspire (manufactured by Perkinelmer). The value after subtracting the background value was calculated as Δabs. The results are shown in Table 21.

TABLE 21

As shown in table 21, decrease in absorbance of CD63 was observed by siRNA treatment of RAB27B, NFE2L2, TLR 2.

While not wishing to be bound by theory, the mesenchymal stem cells prepared by the method of the present invention are characterized by having the following molecular mechanism:

interaction of ∈mesenchymal Stem cells with the substrate used in the present invention

Activation of TLR2 Signal

Activation of the NF- κB Signal and NFE2L2 (NRF 2) Signal

Expression of PGE2 gene and TSG6 gene associated with activation of NF- κB signal is enhanced (high functionalization of MSC)

Expression of RAB27B Gene with activation of NFE2L2 Signal is increased (secretion amount of extracellular vesicles of MSC is increased)

Preparation example 2 preparation of aqueous dispersion containing vitronectin-supported chitin nanofibers

An aqueous dispersion of 2 mass% chitin nanofibers prepared as described in International publication No. 2015/111686 was autoclaved at 121℃for 20 minutes. Then, this aqueous dispersion was mixed and suspended in sterile distilled water (tsukamurella distilled water, manufactured by tsukamurella pharmaceutical factory, ltd.) so as to be 1% (w/v), whereby a sterile aqueous dispersion containing chitin nanofibers was produced. To 1% (w/v) of an aqueous dispersion of chitin nanofibers (5 mL), an aqueous solution (Gibco Vitronectin (VTN-N) recombinant human protein, manufactured by Thermo FISHER SCIENTIFIC Co.) (0.5 mL) containing 500. Mu.g/mL of vitronectin was added, and after mixing by blowing, the mixture was allowed to stand at 4℃for storage overnight, thereby producing an aqueous dispersion of chitin nanofibers carrying vitronectin. (in this specification, the vitronectin-supported chitin nanofibers produced herein are sometimes referred to simply as "base material of production example 2", "production example 2" or "base material 2")

Test example 19 study 1 of culture and passage method of mesenchymal Stem cells by substrate 2 and stirring

For mesenchymal stem cells derived from human umbilical cord (PromoCell, co., # C-12971), mesenchymal stem cell proliferation medium 2 (PromoCell, co., # C-28009) was used, and the culture was performed on a 10cm dish (Corning, co., # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a culture medium composition prepared by adding the substrate of preparation example 2 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.01% (w/v) at an inoculation concentration of 1.5X10- ⁴ cells/mL. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used to perform continuous stirring at 25rpm while culturing in a CO ₂ incubator (37 ℃, 5% or 10% CO ₂) for 3 days. On the 3 rd day of cultivation, cultivation was again performed under the following conditions 1,2, or 3, and observation with a microscope based on fluorescent staining was performed, and proliferation was evaluated.

(Condition 1: additional passage based on substrate 2)

3ML of the culture suspension on day 3 of the culture was separated, and 27mL of a medium composition prepared by adding the substrate of preparation example 2 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.01 (w/v) was added. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used to perform continuous stirring at 25rpm while culturing in a CO ₂ incubator (37 ℃, 5% or 10% CO ₂) for 4 days.

(Condition 2: additional passage based on physical treatment and substrate 2)

An autoclave-sterilized PP straight-through joint (manufactured by Isis, inc. # VRFC 6) was connected to a 30mL syringe (manufactured by Nipro, inc. # 8955) filled with the entire amount of the culture suspension on day 3 of culture, and a new 30mL syringe (manufactured by Nipro, inc. # 8955) was connected to the other end. The extrusion operation from the filling syringe to the empty syringe side was repeated 3 times at about 1mL/s to provide physical shear force, thereby dispersing the spheroids. 3mL of the obtained suspension was separated, and 27mL of a medium composition obtained by adding the substrate of preparation example 2 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.01% (w/v) was added. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used, followed by continuous stirring at 25rpm, while culturing in a CO ₂ incubator (37 ℃, 5% or 10% CO ₂) for 3 days.

( Condition 3: cultivation without addition of substrate 2 (comparative example) )

3ML of the culture suspension on day 3 of the culture was separated, and 27mL of mesenchymal stem cell proliferation medium 2 was added. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) was used, and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) was used to perform continuous stirring at 25rpm while culturing in a CO ₂ incubator (37 ℃, 5% or 10% CO ₂) for 4 days.

(Microscopic observation based on fluorescent staining)

On day 4 of the re-culture, 0.5mL of the culture medium in uniform suspension was collected into a 1.5mL tube, and after centrifugation (600 Xg, 3 minutes), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (600 Xg, 3 minutes). 10. Mu.L of a solution of Calcein-AM (manufactured by Tokugaku Co., # C326) dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 5mL of D-PBS (-) (manufactured by Fuji photo-pure chemical Co., # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 30 minutes. Then, a bright field image and a cell-specific fluorescent staining image were obtained using a fluorescent microscope (manufactured by KEYENCE Co., ltd., BIOR EVO BZ-9000). The results are shown in FIG. 22.

As shown in fig. 22, under both conditions 1 and 2, the proliferated cells were also observed on the newly added substrates. Especially under condition 2, it was revealed that the scattered cells proliferated while wrapping the new substrate, and thus the number of spheroids was also increased.

(Calculation of proliferation Rate)

On days 0, 3 and 0, 4 of culture, 0.5mL of the uniformly suspended culture medium was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added to each, the mixture was stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 150. Mu.L of each was dispensed to a white 96-well plate, luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value (ATP measurement, luminescence intensity) on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 22.

TABLE 22

Proliferation rate	Day 0	Day 3	Passage day 0	Passage day 4
					Condition 1	1	5.1	1	9.4
Condition 2	1	4.5	1	16.6
					Condition 3	1	5.1	1	4.8

As shown in table 22, it was confirmed that the most excellent proliferation was exhibited under the condition of subculturing the substrate 2 added to the material obtained by physically dispersing the spheroids, compared with the condition 3 in which the simple spheroids themselves were continuously partially proliferated. In addition, the culture medium showed a certain proliferation even under condition 1 in which subculture was performed by adding only the substrate 2.

Study 2 of [ test example 20 ] passage method

(Substrate of production example 1 (substrate 1))

For mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used, and the cells were subjected to adhesion culture on a 15em dish (Corning, inc. # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 100mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.05% (w/v) at an inoculation concentration of 1.5X10. 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 50rpm for 6 days. As the culture vessel, a 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. After sterilizing (121 ℃ C. For 20 minutes) a 1L culture glass tank (manufactured by ABLE Corporation) on which triangular blades are mounted by an autoclave, mesenchymal stem cell proliferation medium 2 and penicillin-streptomycin solution (x 100) (manufactured by Fuji photo-pure chemical Co., # 168-23191) were added, and the mixture was set in a BCP-type animal cell culture apparatus (manufactured by ABLE Corporation) to adjust the medium at 37 ℃ C. And 30rpm under the control of compressed air 140ccm and CO ₂ added appropriately so as to be pH 7.5.

(Substrate of production example 2 (substrate 2))

Mesenchymal stem cells derived from human adipose tissue, which were cultured and isolated by the same method as described in the substrate of preparation example 1, were added to a culture medium previously adjusted so that the final concentration of the substrate of preparation example 2 became 0.01% (w/v) at an seeding concentration of 1.5X10 ⁴ cells/mL. The total amount of medium was 1000mL. The culture was performed under the same control conditions as the adjustment. The culture was performed for 6 days, and the culture vessel was left to stand for 10 minutes on the 4 th day of the culture, and half of the culture supernatant was subjected to medium exchange.

(Passage)

For the cells at the time point of the 6 th day of the culture in preparation examples 1 or 2,3 kinds of operations were performed, and the culture was continued. Specifically, cells subjected to a specific operation (operation 1 to operation 3) were added to 30mL of the culture medium composition obtained by adding the substrate of preparation example 1 to the mesenchymal stem cell growth medium 2 so that the final concentration became 0.05% (w/v) or the substrate of preparation example 2 was 0.01% (w/v), and cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 50rpm for 6 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. The culture vessel was allowed to stand for 10 minutes on day 4 of the culture, and half of the culture supernatant was subjected to medium exchange.

Cells receiving procedure 1: 10mL of the cell suspension was separated into a centrifuge tube, and after 10 minutes of standing, the supernatant was removed.

Cells receiving procedure 2: 10mL of the cell suspension was separated in a centrifuge tube, and after centrifugation (300 Xg, 3 min, decel mode), the supernatant was removed. Then, 9773. Mu.L of D-PBS (-) and 277. Mu.L of an enzyme solution prepared by dissolving 35mg of Liberase MNP-S (manufactured by CustomBiotech Co., # 05578566001) in 14mL of D-PBS (-) were added, and incubated in a water bath at 37℃for 30 minutes, and 20 puffs were performed every 10 minutes. Then, 10mL of mesenchymal stem cell proliferation medium 2 was added, and after centrifugation (300×g,3 minutes, decel mode), the supernatant was removed to obtain cells.

Cells receiving procedure 3: after passing 100mL of the cell suspension through a cell sieve (pluriSelect, manufactured by Oreg., # 43-50200-03), 50mL of D-PBS (-) (manufactured by Fuji film and Wako pure chemical industries, ltd., # 045-29795) was added, the net was washed, the net was turned upside down, and the net was washed with an appropriate amount of D-PBS (-), whereby the net-captured spheres were collected, centrifuged (300 Xg, 3 minutes, decel mode), and the supernatant was removed. Then, 9723. Mu.L of D-PBS (-) and 277. Mu.L of an enzyme solution prepared by dissolving 35mg of Liberase MNP-S (manufactured by CustomBiotech Co., # 05578566001) in 14mL of D-PBS (-) were added, and incubated in a water bath at 37℃for 30 minutes, and 20 puffs were performed every 10 minutes. Then, 10mL of the mesenchymal stem cell growth medium 2 was added and passed through a cell sieve (manufactured by Nissan chemical Co., ltd.) having a pore size of 65 μm using a syringe, to obtain a filtrate containing single cells from which the substrate had been removed. After centrifugation (300 Xg, 3 min, decel mode), the supernatant was removed, 10mL of mesenchymal stem cell proliferation medium 2 was added, and the cell concentration was measured by using a cell counter (manufactured by BIO-RAD Co., ltd., TC-20) to obtain 4.5X10: 10 ⁵ cells.

(Cell staining)

The culture medium inoculated after uniform suspension (day 0 of culture) and 1mL of the culture medium at day 6 of culture were collected in a 1.5mL tube, and after centrifugation (300 Xg, 3 min, decel mode), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (300 Xg, 3 min, decel mode). 20. Mu.L of Calcein-AM (manufactured by Tonka Chemie, # C326) solution dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 10mL of D-PBS (-) (manufactured by Fuji photo-pure Co., ltd. # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 15 minutes. Then, a bright field image and a living cell-specific fluorescent staining image were obtained using EVOS (registered trademark) FL Auto (manufactured by thermo fisher). The results are shown in FIG. 23. The scale bar represents 1000. Mu.m.

As shown in fig. 23, in the cells receiving operations 2 and 3 on day 0, no spheres were observed, but spheres were observed on day 6. In addition, in the cells that received operation 1, since only a new substrate was added to the spheres, spheres were also observed on day 0, but on day 6, sparse spheres with low fluorescence intensity appeared, and a state where the cells moved from the formed spheres to the newly added substrate was observed. From the above results, it was confirmed that in any of the substrates 1 and 2, the cells can be efficiently passaged not only by a method of inoculating the single cells into a fresh medium containing a new substrate after once single-cell formation of the spheres, but also by a method of adding a fresh medium containing a substrate to the formed spheres.

[ Preparation example 3]

An autoclave sterilization treatment was performed for a2 mass% aqueous dispersion of chitosan nanofibers prepared as described in International publication No. 2015/111686 at 121℃for 20 minutes. Then, the aqueous dispersion was mixed and suspended in sterile distilled water (tsukamurella distilled water, manufactured by tsukamurella pharmaceutical factory, ltd.) so as to be 1% (w/v), thereby producing a sterile aqueous dispersion containing chitosan nanofibers. (in this specification, the chitosan nanofibers produced herein are sometimes simply referred to as "substrate of production example 3", "production example 3" or "substrate 3")

Test example 21 comparison of combinations of substrates and stirring conditions

For mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used, and the cells were subjected to adhesion culture on a 15cm dish (Corning, inc. # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL of a medium composition containing the substrate of preparation example 1 at a final concentration of 0.05% (w/v), the substrate of preparation example 2 at a final concentration of 0.01% (w/v), the substrate of preparation example 3 at a final concentration of 0.04% (w/v), or the cells were added to 30mL of a medium containing no substrate at an inoculation concentration of 1.5X10. ⁴ cells/mL, and the medium was cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 50rpm for 6 or 7 days. As a culture vessel, a 30mL disposable reactor (manufactured by ABLE Corporation, # BWV-S03A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. Cells were added to 5mL of the culture medium composition prepared in the same manner, and cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under static conditions for 7 days using EZ-BindShut (registered trademark) SP (low adhesion surface) 6 well plate (manufactured by AGC Techno Glass Co., #4810-800 SP) as a culture vessel. On day 4 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange.

(Cell staining)

1ML of the culture medium in uniform suspension was collected into a 1.5mL tube, and after centrifugation (300 Xg, 3 minutes, decel mode), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (300 Xg, 3 min, decel mode). 20. Mu.L of Calcein-AM (manufactured by Tonka Chemie, # C326) solution dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 10mL of D-PBS (-) (manufactured by Fuji photo-pure Co., ltd. # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 15 minutes. Then, a bright field image and a living cell-specific fluorescent staining image were obtained using EVOS (registered trademark) FL Auto (manufactured by thermo fisher). The results are shown in FIG. 24. The scale bar represents 1000. Mu.m.

As shown in fig. 24, when the substrate 1 and the substrate 2 were used for suspension culture under stirring, a spherical body with a clear outline was obtained. On the other hand, in the case of using the base material 3, a large number of spheres smaller than in the case of using the base material 1 or the base material 2 were observed. In addition, in the case where the base material is not used, substantially no sphere is formed. In addition, when the base material 2 is used under the standing condition, a large aggregate is formed, and the dispersibility of the spheres is reduced as compared with the case of using the base material 1 under the standing condition. From the above results, it was revealed that the culture using the base material of production example 1 or production example 2 was effective under stirring in order to obtain spheres having uniform sizes.

(Calculation of proliferation Rate)

On day 0, 4, 6 or 7 of the culture, 0.5mL of the uniformly suspended culture medium was collected, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability assay, manufactured by Promega corporation) was added thereto, stirred by a vortex mixer, allowed to stand at room temperature for 10 minutes, 100. Mu.L of each of the white 96-well plates was dispensed, luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and luminescence value of the medium itself was subtracted to determine the number of living cells. The relative value at which the RLU value on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 23. The symbol "-" in the table indicates that measurement was not performed.

TABLE 23

As shown in table 23, the cell number increased with time in all the substrates, and the substrates 1 and 2 showed a high proliferation rate on day 7 under stirring compared to the rest condition. In addition, under stirring conditions, the substrate 2 has a higher proliferation rate than the substrate 1. On the other hand, in the case where the substrate is not used, the cells do not proliferate.

Test example 22 enlarged study

For mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used to carry out an adherent culture on a 15cm dish (Corning, inc. # 430167) for 3 days. Then, the cells were peeled off using DETACHKIT (manufactured by PromoCell Co., #C-41210). After sterilizing (121 ℃ C. For 20 minutes) a 1L culture glass tank (manufactured by ABLE Corporation) equipped with triangular blades using an autoclave, mesenchymal stem cell proliferation medium 2 and penicillin-streptomycin solution (x 100) (manufactured by Fuji film and Wako pure chemical industries, ltd. # 168-23191) were added, and the mixture was placed in a BCP-type animal cell culture apparatus (manufactured by ABLE Corporation) under the control of compressed air 140ccm and CO ₂ was appropriately added so as to be pH7.5, The medium was adjusted at 37℃and 30rpm for 30 minutes. The detached cells were added to a culture medium adjusted in advance so that the final concentration of the substrate of production example 1 became 0.05% (w/v) or the final concentration of the substrate of production example 2 became 0.01% (w/v) at an inoculation concentration of 1.5X10 ⁴ cells/mL, and the total amount of the culture medium was 1000mL. The culture was performed under the same control conditions as the adjustment. The detached cells were added to 100mL of a culture medium composition containing the substrate of preparation example 1 at a final concentration of 0.05% (w/v) or the substrate of preparation example 2 at a final concentration of 0.01% (w/v) in a mesenchymal stem cell growth medium 2 at an inoculation concentration of 1.5X10- ⁴ cells/mL, and the mixture was stirred at 50rpm in a CO ₂ incubator (37 ℃ C., 3 ℃ C.), 5% CO ₂) in the culture. As the culture vessel, a 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. In the case of using the substrate of preparation example 1, the culture was performed for 7 days, and the culture vessel was left to stand for 10 minutes on the 4 th day of the culture, and half of the culture supernatant was subjected to medium exchange. In the case of using the substrate of preparation example 2, 4 days of cultivation was performed.

(Calculation of proliferation Rate)

When the substrate of preparation example 1 was used, 0.5mL of the uniformly suspended culture solution was collected on days 0, 4 and 7 of the culture, and when the substrate of preparation example 2 was used, 0.5mL of the uniformly suspended culture solution was collected on days 0 and 4 of the culture, 0.5mL of ATP reagent (CellTiter-Glo (registered trademark) was added to the culture solution, cell viability was measured by the luminescence method, promega corporation), the mixture was stirred by a vortex mixer, and after standing at room temperature for 10 minutes, 100. Mu.L of each of the culture solutions was dispensed into a white 96-well plate, and the luminescence intensity (RLU value) was measured by Enspire (PERKIN ELMER corporation) and the luminescence value of the culture medium itself was subtracted to measure the number of living cells. The relative value at which the RLU value on day 0 of culture was set to 1 was used as the cell proliferation rate. The results are shown in Table 24.

TABLE 24

As shown in table 24, the proliferation rates equal to or higher than 100mL were obtained on a 1L scale on any substrate. From the above results, it was revealed that the expansion was possible while maintaining the efficiency.

(Cell staining)

1ML of the culture medium in uniform suspension was collected into a 1.5mL tube, and after centrifugation (300 Xg, 3 minutes, decel mode), the culture supernatant was removed. Cells were suspended in 1mL of D-PBS (-) (Fuji photo-pure Co., # 045-29795), and the culture supernatant was removed after centrifugation (300 Xg, 3 min, decel mode). 20. Mu.L of Calcein-AM (manufactured by Tonka Chemie, # C326) solution dissolved in DMSO at a final concentration of 0.5mg/mL was dissolved in 10mL of D-PBS (-) (manufactured by Fuji photo-pure Co., ltd. # 045-29795) as a staining solution. Cells were suspended in 1mL of the staining solution, transferred to a 12-well plate (manufactured by Corning Co., # 351143), and incubated in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 15 minutes. Then, a bright field image and a living cell-specific fluorescent staining image were obtained using EVOS (registered trademark) FL Auto (manufactured by thermo fisher). The results are shown in FIG. 25. The scale bar represents 1000. Mu.m.

As shown in fig. 25, spheres equivalent to 100mL were obtained on a 1L scale on any base material.

Test example 23 observation of spherical section

For mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used to carry out an adherent culture on a 15cm dish (Corning, inc. # 430167) for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 30mL or 100mL of a culture medium composition in which the substrate of preparation example 1 was added to the mesenchymal stem cell growth medium 2 so that the final concentration became 0.05% (w/v) or the substrate of preparation example 2 was added so that the final concentration became 0.01% (w/v) at an inoculation concentration of 1.5X10. 10 ⁴ cells/mL, culturing was performed in a CO ₂ incubator (37 ℃, 5% CO ₂) for 4 or 7 days with stirring at 50 rpm. As the culture vessel, 30mL (manufactured by ABLE Corporation, # BWV-S03A) or 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. As a comparative example, the isolated cells were added to the mesenchymal stem cell propagation medium 2, inoculated at 1.5X10- ⁴ cells/well/200. Mu.L on PrimeSurface (registered trademark) plate 96U (manufactured by SUMITOMO BAKELITE Co., #MS-9096U), and cultured in a CO ₂ incubator (37 ℃ C., 3. F), 5% CO ₂) for 4 days. The spheres obtained using the substrate of preparation example 1 were collected by passing the culture solution through a cell sieve (pluriSelect, inc. # 43-50200-03) having a pore size of 200 μm on day 7 of the culture, washing the cell sieve with D-PBS (-), turning the net upside down, and collecting the spheres captured on the net using D-PBS (-), thereby obtaining spheres. As the spheres obtained using the substrate of preparation example 2 and spheres obtained under the conditions of comparative example, spheres on day 4 of culture were used.

(Preparation of frozen section)

After centrifugation of the spheres (200 Xg, 3 minutes), the supernatant was removed, suspended in 50. Mu.L of mesenchymal stem cell proliferation medium 2, and cooled on ice. The spheres were colloidized according to the product instructions of iPGell (manufactured by GENOSTAFF Co., # PG 20-1). Specifically, 10. Mu.L of A-solution cooled with ice was added to the sphere suspension, and after sufficient blowing, 50. Mu.L of B-solution at room temperature was added, and immediately blown 3 times. After the sample tube was left standing at room temperature for 1 minute, it was confirmed that the sample tube coagulated into a gel. Then, 4% paraformaldehyde/phosphate buffer (manufactured by Fuji photo-pure chemical Co., # 163-20145) was added thereto, and the mixture was incubated overnight, thereby immobilizing the mixture. Then, the solution was washed with D-PBS (-), and sucrose (manufactured by Fuji photo-pure Co., ltd., # 196-00015) was dissolved in D-PBS (-) so that the final concentration became 10 or 20 or 30% (w/v), immersed in 10% sucrose PBS solution for 4 hours, immersed in 20% sucrose PBS solution overnight, and immersed in 30% sucrose PBS solution overnight. Then, the gelled spheres were placed in a plastic embedding vessel (manufactured by Sakura Finetech Japan, # 4730) containing a frozen embedding medium (manufactured by Leica Microsystems, inc. # 3801480), and cooled to-100℃in hexane/isopentane 1 using a bench-type cooling trap (manufactured by Tokyo instruments, UT-2000): 1 in solution. The frozen embedded block was thinly cut into 10 to 30 μm using a cryostat (CM 3050S, manufactured by Leica Microsystems Co., ltd.) and attached to a glass slide (Songbo Nitro Co., ltd. # S7445). The embedding medium on the slide glass was removed by running water, immersed in hematoxylin (Sakura Finetek Japan Co., # 6187-4P) at room temperature for 5 minutes, and then washed with running water for 5 minutes. Then, the mixture was dehydrated and transparent according to a usual method, and mounted with a cover slip (manufactured by Songbo Nitro Co., ltd. # C024321) and a mounting agent (manufactured by FALMA Co. # 308-600-1), and observed with an inverted microscope (manufactured by Olympus Corporation Co. # IX 73). The acquired image is shown in fig. 26. The scale bar indicates 100. Mu.m.

As shown in fig. 26, in the comparative example, the nuclei were stained to the inside of the spheres, but in the spheres of the substrates 1 and 2, the inside of the spheres was not stained. From the above results, it was revealed that the spheres obtained using the base material of preparation examples 1 or 2 could be in a state of being internally packed with the base material. In the case of the sphere in which cells are tightly packed into the interior as in the comparative example, it is considered that nutrients, oxygen, etc. outside cannot reach the center of the sphere, and the cells in the center of the sphere may die. On the other hand, in the sphere produced by the method of the present invention, the substrate is wrapped inside the sphere, so that the distance from the surface to the center of the sphere is long, and the number of cells that are difficult to reach, such as nutrients and oxygen, can be reduced. Therefore, according to the present invention, the cells can be more efficiently proliferated.

Test example 24 application of mesenchymal Stem cells prepared by the method of the present invention to deformed knee arthropathy

(Preparation of adhesion cultured cells (2D group))

In the case of mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used, and the cells were inoculated onto a 15cm dish (Corning, inc. # 430167) at an inoculation density of 5000 cells/cm ², and subjected to an adhesion culture for 3 days. Then, cells were peeled off using DETACHKIT (manufactured by PromoCell Co., #C-41210), and cell concentrations were counted using a cell counter (manufactured by BIO-RAD Co., #TC-20). The desired cell number was collected into a new centrifuge tube, centrifuged (200×g,3 minutes), and the supernatant was removed, and the tube was suspended in STEMCELLBANKER GMP grade (manufactured by the whole pharmaceutical industry Co., ltd. # CB 045) to give 5×10 ⁶ cells/tube (visual), stored at-80℃in CoolCell LX (manufactured by Corning Co. # 432002), and stored in liquid nitrogen the next day.

(Preparation of stirred culture cells (3D group))

In the case of mesenchymal stem cells derived from human adipose tissue (CellSource, inc. # 0111201), mesenchymal stem cell proliferation medium 2 (PromoCell, inc. # C-28009) was used, and the cells were inoculated onto a 15cm dish (Corning, inc. # 430167) at an inoculation density of 5000 cells/cm ², and subjected to an adhesion culture for 3 days. Then, cells were detached using DETACHKIT (manufactured by PromoCell Co., #C-41210), and the cells were added to 100mL of a culture medium composition prepared by adding the substrate of preparation example 1 to mesenchymal stem cell proliferation medium 2 so that the final concentration became 0.05 (w/v) at an inoculation concentration of 1.5X10. 10 ⁴ cells/mL, and culturing was performed in a CO ₂ incubator (37 ℃ C., 5% CO ₂) under stirring at 50rpm for 7 days. As the culture vessel, a 100mL disposable reactor (manufactured by ABLE Corporation, # BWV-S10A) and a dedicated magnetic stirrer (manufactured by ABLE Corporation, # BWS-S03N 0S-6) were used. On day 4 of the culture, the culture vessel was left to stand for 10 minutes, and half of the culture supernatant was subjected to medium exchange.

(Separation of spheres)

On day 7 of the culture, the whole amount of the culture solution was passed through a cell sieve (pluriSelect, inc. # 43-50200-03) having a pore size of 200 μm, and then washed with 100mL of D-PBS (-), the net was turned upside down, and the spheres trapped on the net were recovered with 50mL of D-PBS (-). Then, the spheres were precipitated by natural sedimentation, and the supernatant was removed as 10mL of cell suspension.

(Enzyme treatment)

A solution of 554. Mu.L of Liberase (registered trademark) TM RESEARCH GRADE (manufactured by Merck Co., # 5401119001) dissolved in D-PBS (-) at a final concentration of 13U/mL, 2mL of TrypLE (registered trademark) Select Enzyme (10X) free of phenol red (manufactured by Thermo Fisher Co., # A1217701), and 7446. Mu.L of D-PBS (-) were mixed to prepare 10mL of an Enzyme solution. The enzyme solution heated to 37℃was added to the cell suspension, and transferred to a cell dispersion tool (manufactured by ABLE Corporation). The cell dispersion tool was set in a high-rotation stirrer (made by ABLE Corporation) with a temperature adjusting function for the dispersion tool, which had been heated to 37℃and the cells were dispersed at 1200rpm for 20 minutes.

(Purification, cryopreservation)

Using Eppendorf Research (registered trademark) plus100 to 1000. Mu.L (manufactured by Eppendorf Co., # 3120000062) with a discharge amount of 1mL, 50 times of blowing was performed, 20mL of mesenchymal stem cell proliferation medium 2 was added, the enzyme was neutralized, the cell suspension was filled into a 100mL syringe, the substrate was removed by passing through a cell screen (manufactured by Nissan chemical Co., ltd.) with a pore size of 65 μm, a filtrate containing single cells was obtained, and the cell concentration was counted by using a cell counter (manufactured by BIO-RAD Co., # TC-20). The desired cell number was collected into a new centrifuge tube, centrifuged (200×g, 3 minutes), and the supernatant was removed, suspended in STEMCELLBANKER GMP grade (manufactured by the whole pharmaceutical industry Co., ltd. # CB 045) so as to be 5×10 ⁶ cells/tube, stored at-80℃in CoolCell LX (manufactured by Corning Co., ltd. # 432002), and stored in liquid nitrogen the next day.

(Quantification of residual amount of substrate in 3D group)

3X 10 ⁵、1×10⁶、1×10⁷ cells were separated from the purified cell suspension, and after centrifugation (300 Xg, 3 min, decel mode), the culture supernatant was removed, and the cells were dissolved in 1mL Reagent A100 (manufactured by chemometec Co., # 910-0003) by addition and suspension. Next, the residual substrate contained in the Cell suspension was quantified using Chitosan Assay Kit (manufactured by Cell Biolabs, # XAN-5126). After 1mL of the cell lysate was centrifuged (12300×g,3 minutes), 0.9mL of the supernatant was removed, and 0.4mL of a 25M aqueous sodium hydroxide solution was added thereto, and the mixture was heated at 121 ℃ for 3 hours, thereby performing deacetylation treatment. Then, 1mL of ethanol was added, and the mixture was vortexed (2500 speed, 1 minute) to precipitate a polymer, and after centrifugation (12300×g,3 minutes) and supernatant removal (1000 μl) were repeated 2 times, 1mL of water was further added, and then (12300×g,3 minutes) and supernatant removal (1300 μl) were performed, whereby sodium hydroxide was removed, and the sample was dried and solidified by drying under reduced pressure. To the dried sample, 0.2mL of an acetic acid buffer for measurement was added to dissolve the sample, and the sample subjected to the pretreatment for measurement was dispensed into a transparent bottom side white 96-well plate (manufactured by Corning Co. # 3632) in an amount of 250. Mu.L per 1 well according to the above Kit specification, and absorbance at 540nm was measured using an enzyme-labeled instrument (manufactured by Tecan Co. # INFINITEM PRO). The concentration of the substrate contained in the sample was calculated by using a calibration curve prepared by using the standard substance contained in the Kit. Table 25 shows the cell numbers and the amounts of the substrates contained. The correction curve was derived from table 25, and the amounts of the substrates contained in the suspensions of 0.975×10× 10 ⁴ and 7.5×10 ⁵ cells were estimated using the correction curve (table 26).

TABLE 25

Cell count	Liquid amount	Measurement value	Containing the amount of the base material
				(Individual cells)	(mL)	(ug/mL)	(ug)
3x10^5	0.2	0.56	2.78
				1x10^6	0.2	0.99	4.95
1×10^7	0.2	1.63	8.14

(Correction Curve type)

Y: amount of substrate (μg)

X: cell number (. Times.10 ⁵ cells)

y＝0.0469x+3.5253

TABLE 26

Cell count	Estimating the amount of the substrate
		(Individual cells)	(ug)
7.5×10^5	3.88

(Acquisition of PGE2 production amount measurement sample)

In the case of group 2D, 1mL of mesenchymal stem cell proliferation medium 2 was inoculated at an inoculation density of 4000 cells/em ² into a 24-well plate (manufactured by Corning Co., # 3526), cultured for 4 days, the supernatant was removed, washed with D-PBS (-), 1mL of the mesenchymal stem cell proliferation medium 2 or the mesenchymal stem cell proliferation medium 2 containing TNF-alpha (manufactured by R & D Systems Co., # 210-TA) at a final concentration of 20ng/mL was added, The culture was carried out in a CO ₂ incubator (37 ℃ C., 5% CO ₂) for 24 hours, and the culture supernatant thus obtained was used as a PGE2 measurement sample and the cells were used as ATP measurement samples. For the 3D group, 1.2mL of the culture medium uniformly suspended on day 7 of the culture was collected into a 1.5mL tube, and after centrifugation (300 Xg, 3min, decel mode), the culture supernatant was removed. Then, the cells were dissolved by adding and suspending them in 1.2mL Reagent A100 (chemometec, manufactured by chemometec, # 910-0003), 100. Mu.L of Reagent B (chemometec, manufactured by # 910-0002) was added to the 100. Mu.L-1.5 mL tube, and the mixture was loaded onto Vial-Cassette (registered trademark) (chemometec, manufactured by # 941-0012) and then the cell concentration was counted using Nucleocounter NC-200 (registered trademark) (chemometec). 1X 10 ⁵ cells were collected into a centrifuge tube, centrifuged (300 Xg, 3 min, decel mode), the supernatant was removed, washed with D-PBS (-), 1mL of mesenchymal stem cell proliferation medium 2 or mesenchymal stem cell proliferation medium 2 containing TNF- α (manufactured by R & D Systems Co., # 210-TA) at a final concentration of 20ng/mL was added, and the mixture was subjected to 24-well ultra-low adhesion surface plate (manufactured by Corning Co.), # 3473) was cultured in a CO ₂ incubator (37 ℃ C., 5% CO ₂) in a stationary state for 24 hours. after the incubation, a supernatant was obtained by centrifugation (300×g, 3min, decel mode) and used as a PGE2 measurement sample, and cells were used as ATP measurement samples.

(Cell count calculation and PGE2 production measurement)

To cells, 1mL of an ATP reagent (CellTiter-Glo (registered trademark) luminescence method cell viability assay, manufactured by Promega corporation) was added, and after allowing the cells to stand at room temperature for 10 minutes by blowing, 150 μl of each was dispensed into a white 96-well plate, and luminescence intensity (RLU value) was measured by Enspire (manufactured by PERKIN ELMER corporation), and the luminescence value of the medium itself was subtracted to determine ATP value, which was used as the number of living cells. Next, PGE2 contained in the recovered culture supernatant was quantified using PGE2 ELISA kit (EnzoLife Science, inc. # ADI-900-001). 100. Mu.L of standard solution (standard) diluted with Assay Buffer and culture supernatant were added to each well of a 96-well plate attached to the kit. Next, 50 μl of blue conjugate was added to each well. mu.L of yellow anti-ibody was further added to each well and shaken at room temperature for 2 hours. Next, the solution was discarded, and after 400. Mu.L/well of the washing liquid was added thereto, the solution was discarded. The above operation was repeated 3 times. 200. Mu.L of pNpp base solution was added to each well and shaken for 45 minutes at room temperature. Finally, 50. Mu.L of a stop solution was added to stop the reaction, and absorbance at 405nm was measured. The PGE2 concentration contained in each sample was calculated by four-parameter logistic regression of the calibration curve. To calculate the secretion per unit cell number, the calculated PGE2 amount is divided by the ATP value to calculate the relative value. The results are shown in Table 27.

TABLE 27

As shown in table 27, it was confirmed that the secretion amount of PGE2 was increased in the 3D group as compared with the 2D group. According to the above results, it was shown that the mesenchymal stem cells prepared using the method of the present invention have a higher anti-inflammatory effect than the mesenchymal stem cells prepared by the previous adhesion culture.

(Analysis of cell surface markers)

After washing the single-cell-state cells of the 2D and 3D groups with a washing buffer (D-PBS (-) containing 2% FBS), BV421 Mouse Anti-Human CD73 (manufactured by BD Co., # 562430), APC Mouse Anti-Human CD90 (manufactured by BD Co., # 559869), BV650 Mouse Anti-Human CD105 (manufactured by BD Co., # 563466), FITC Anti-CD11b antibody [ M1/70] (manufactured by abcam Co., # ab 24874), and PE Mouse Anti-HumanCD (manufactured by BD Co., # 555822) were added, respectively, and incubated on ice and protected from light for 30 minutes. BV421 Mouse IgG1, k Isotype Control (manufactured by BD Co., # 562438), APC Mouse IgG1, kappa Isotype Control (manufactured by BD Co., # 555751), BV650 Mouse IgG1, k Isotype Control (manufactured by BD Co., # 563231), FITC RAT IGG b, kappa monoclone [ eB149/10H5] -Isotype control (manufactured by abcam Co., # ab 136125), PE Mouse IgG1, kappa Isotype Control (manufactured by BD Co., # 555749) were added to the negative control, respectively. The incubated cells were washed 2 times with a washing buffer, and after treatment with a 35 μm cell sieve, the positive rate of each cell surface marker was calculated by measurement using BD LSRFortessa (registered trademark) X-20 (manufactured by BD Co.). CD73, CD90 and CD105 are positive markers of mesenchymal stem cells, and CD11b and CD34 are negative markers of mesenchymal stem cells. The results are shown in Table 28.

TABLE 28

As shown in table 28, both the cells of the 2D group and the 3D group expressed CD73, CD90, CD105, and neither expressed CD11b, CD34 as a negative marker. That is, it was found that these cells maintained the state of mesenchymal stem cells.

(Preparation of deformable gonarthrosis rat)

Made using 7 week old male Wistar rats (Japan SLC). 3 kinds of mixed anesthetic solutions (1.8 mL/kg) were subcutaneously administered to rats to be anesthetized. For the final dose of each narcotic, the midazolam was 2mg/kg, medetomidine hydrochloride was 0.4mg/kg, and butorphanol tartrate was 5mg/kg. Simultaneously with anesthesia, an analgesic (carprofen 5mg/kg, 1 mL/kg) was subcutaneously administered, and pain was treated for postoperative pain. The right hind limb of the rat under general anesthesia was then shaved and the skin on the inside of the patella was cut longitudinally. Then, the muscle tissue is incised to expose the medial collateral ligament. The medial collateral ligament and the anterior cruciate ligament were cut by the MANI (registered trademark) ophtalmic Knife (MANI corporation, strain 22.5 °), and the meniscus was cut from the femur and tibia, and the meniscus was removed. Suturing with suture thread or surgical stapler, and then dripping iodine tincture into the suture part for disinfection. The two mixed antagonists (1 mL/kg) were subcutaneously administered and awakened from anesthesia. With respect to the final amount of antagonist, the atemezole hydrochloride was 1.2mg/kg, and flumazenil was 0.01mg/kg. After the animals were awake, it was confirmed whether or not there was abnormality in the normal state. For sham rats, suturing and sterilization were performed after incision of the knee skin.

(Administration of mesenchymal Stem cells prepared using the method of the present invention to a rat suffering from a knee joint disease with deformability)

Rats were grouped as described in table 29 below. After 3 days from the operation, rats were anesthetized with 1.5 to 3.0% isoflurane, and then mesenchymal stem cells or a hyaluronic acid preparation (Suvenyl Dispo Joint Injection mg, manufactured by Zhongwei pharmaceutical company, positive control) was administered into the right hind limb knee joint of each rat. For mesenchymal stem cells, 1 time after 3 days of surgery, and for hyaluronic acid preparations, a total of 4 times after 3, 10, 17, and 24 days of surgery, 50 μl each, were administered. The mesenchymal stem cells were concentration-adjusted using physiological saline (OTSUKA NORMAL SALINE, manufactured by tsukamu pharmaceutical factory Co.) and administered using a 1mL syringe with a 26G needle.

TABLE 29

(Bipedal pressure differential pain assessment)

Bipedal pressure differential pain assessment was performed 27 days after surgery. In order to obtain a result with higher accuracy, the individual number of the evaluation animal is not clearly shown to the evaluator, and the evaluation is performed by blind test. All rats were placed in a rack (holder) for evaluation of bipedal pain, and left standing for several minutes, and then the load of the left and right hind limbs was measured using a bipedal pain measuring device (Bio RESEARCH CENTER Co.). The measurement was performed until 5 times of data could be obtained for every 1. The load balance (R/L load) was calculated, and the average value of the load balance was obtained for every 1. In addition, using tukey test, a significant difference test was performed with respect to the control group, and the p value was calculated. The results are shown in Table 30.

TABLE 30

	False operation group	Control	2D group	3D group	SUVENYL
						Average of	0.99	0.69	0.85	0.91	0.92
SD	0.0279	0.1104	0.0774	0.1074	0.088
						P value	-	-	0.0032	<0.001	<0.001

As shown in table 30, the average value of load balance was significantly increased in any of the cell administration groups as compared to the control group. This means that the administration of cells inhibited pain in rats. In addition, the value according to the average value of the load balance shows that the pain suppressing effect of the 3D group can be higher than that of the 2D group.

Industrial applicability

According to the present invention, adherent cells of good quality can be produced in large quantities with high efficiency. Thus, the present invention is preferably used for the preparation of cells for use in living body transplantation, for example. Therefore, the present invention is extremely useful in the technical field of biological transplantation.

The present application is based on Japanese patent application No. 2021-169860 (application day: 10/15/2021) and Japanese patent application No. 2022-02397 (application day: 2/18/2022), which are filed in Japan, and the contents thereof are all included in the present specification.

Claims

1. A method for culturing adherent cells, comprising a step of culturing adherent cells in suspension in a medium comprising nanofibers composed of water-insoluble polysaccharides, wherein the culture is performed with stirring.

2. The method according to claim 1, wherein the stirring condition is a state in which nanofibers and cells are suspended in a medium, and a state in which the nanofibers and cells are continuously moved in a system by an external force.

3. The method according to claim 1 or 2, wherein the stirring is carried out by means of a device accompanied by stirring blades, the rotational speed of which is 0.01-50.0 m/min in terms of blade tip speed.

4. A method according to any one of claims 1 to 3, wherein the agitation is carried out without interruption during cell culture.

5. The method according to any one of claims 1 to 4, wherein the amount of the nanofibers composed of the water insoluble polysaccharide added to the medium is 0.0001 to 0.2% (w/v).

6. The method according to any one of claims 1 to 5, wherein the nanofibers comprising water insoluble polysaccharides are carried on the extracellular matrix.

7. The method according to any one of claims 1 to 6, wherein the water-insoluble polysaccharide is at least one selected from the group consisting of chitin, cellulose, and hemicellulose.

8. The method of claim 6 or 7, wherein the extracellular matrix is at least one selected from the group consisting of collagen, fibronectin, vitronectin, laminin, RGD sequence, and cadherin.

9. The method of any one of claims 1-8, wherein the adherent cells are selected from the group consisting of stem cells, progenitor cells, adult non-stem cells, primary cultured cells, cell lines, and cancer cells.

10. The method of any one of claims 1-9, wherein the culture medium further comprises chitosan nanofibers.

11. A method for producing spheres of adherent cells having a uniform sphere size, comprising a step of culturing adherent cells in suspension in a medium containing nanofibers composed of water-insoluble polysaccharides, wherein the culture is carried out with stirring.

12. The method of claim 11, wherein the stirring condition is a state in which nanofibers and cells are suspended in a medium, and a state in which the nanofibers and cells are continuously moved in a system by an external force.

13. The method of claim 11 or 12, wherein the stirring is performed by a device accompanied by stirring blades at a rotational speed of 0.01 to 50.0 m/min at the blade tip speed.

14. The method of any one of claims 11-13, wherein the agitation is performed without interruption during cell culture.

15. The method according to any one of claims 11 to 14, wherein the amount of the nanofibers composed of the water insoluble polysaccharide added to the medium is 0.0001 to 0.2% (w/v).

16. A method for separating spheres comprising the step of supplying a suspension of spheres produced by the method according to any one of claims 11 to 15 to a cell screen.

17. A method of unicellular adherent cells in the form of spheres comprising:

18. A mesenchymal stem cell in which the expression of at least one gene selected from the group consisting of CD55, HMOX1, TSPAN7, RAB27B, IL, GPX3, and MFAP4 is increased compared to a mesenchymal stem cell cultured by an adhesion culture.

19. The mesenchymal stem cell of claim 18, wherein production of extracellular vesicles is also promoted compared to mesenchymal stem cells cultured by adherent culture.

20. The mesenchymal stem cell of claim 19, wherein the extracellular vesicle is an exosome.

21. A method for promoting the production of extracellular vesicles of mesenchymal stem cells, which comprises a step of culturing mesenchymal stem cells in suspension in a medium comprising nanofibers composed of a water-insoluble polysaccharide, wherein the culturing is performed with stirring.

22. A method for producing mesenchymal stem cells in which production of extracellular vesicles is promoted, comprising a step of culturing mesenchymal stem cells in suspension in a medium comprising nanofibers composed of water-insoluble polysaccharides, wherein the culture is performed with stirring.

23. The method of claim 21 or 22, wherein the extracellular vesicles are exosomes.

24. A formulation for use in the treatment of inflammatory diseases comprising the mesenchymal stem cells of any one of claims 18 to 20.