Medium composition for suspension culture of adherent cells

Abstract

The present invention provides a medium composition for suspension culture of adhesive cells, comprising: (1) chitin nanofibers; and (2) chitosan nanofibers or polysaccharides.

Description

Technical Field

[0001] The present invention relates to a medium composition for suspension culture of adhesive cells and a method for suspension culture of adhesive cells. More specifically, the present invention relates to a medium composition for suspension culture of adhesive cells comprising chitin nanofibers and chitosan nanofibers and a method for suspension culture of adhesive cells using the medium composition. Background Art

[0002] In the field of regenerative medicine, the construction of organ regeneration methods using pluripotent stem cells such as iPS cells and ES cells has been studied intensively. However, the establishment of ES cells is accompanied by ethical issues, and it is also recognized that iPS cells have the risk of canceration and the long culture time. Therefore, the possibility of using methods such as mesenchymal stem cells with a lower risk of canceration, adult stem cells such as neural stem cells, progenitor cells such as preadipocytes and precardiomyocytes with a shorter differentiation induction time, or chondrocytes is also being explored.

[0003] Treatment methods using adult stem cells require a large amount of high-quality adult stem cells, and therefore require a culture method that can efficiently prepare high-quality cells. For example, it is known that mesenchymal stem cells can be easily proliferated by monolayer culture (also known as two-dimensional (2D) culture, etc.) in a culture dish.

[0004] However, since the number of cells that can be cultured in a culture dish is limited, when using this method to culture cells in large quantities, it is necessary to study the stacking of many culture dishes, etc., so this culture method is not suitable from the perspective of mass production. In addition, in recent reports, the two-dimensional culture method using a culture dish has the possibility of reducing the undifferentiation ability and proliferation ability of mesenchymal stem cells, and the functions such as migration ability, anti-inflammatory effect, etc., including homing, possessed by mesenchymal stem cells are also likely to be reduced. Therefore, the two-dimensional culture method using a culture dish is not suitable from the perspective of producing high-quality cells.

[0005] On the other hand, as the method for cultivating adhesive cells in large quantities, report that cell is cultivated under the state of being adhered to microcarrier and the device (patent documentation 1) that makes its propagation.But, the microcarrier that can obtain usually at present can settle in culture fluid under standing condition, therefore, need to stir during cultivation.Pointed out the problem that collision etc. cause cell death due to this stirring between carrier.In addition, another problem is, when reclaiming cell from microcarrier for the purpose such as replacement culture medium, enlarged culture scale, need to peel off between cell, cell and carrier, and the proteolytic enzymes such as trypsin used in this peeling process can make cell be damaged, cause thus the survival rate of cell to reduce.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 3275411 Summary of the invention

[0009] Problems to be solved by the invention

[0010] In view of the above background, the present invention aims to provide a novel culture method that can solve multiple problems in suspension culture of adherent cells using conventional microcarriers, such as cell death caused by collision of microcarriers during stirring and damage to cells caused by proteolytic enzyme treatment during subculture.

[0011] The inventors of the present application have repeatedly conducted in-depth research to solve the above-mentioned problems. As a result, they found that by adding chitin nanofibers and chitosan nanofibers or specific polysaccharides in a liquid culture medium at a specific ratio, the following can be achieved: (1) The liquid culture medium can culture adhesive cells in suspension, and since stirring is not required, the adhesive cells can be efficiently proliferated; (2) The liquid culture medium can easily redisperse the carrier and the adhesive cells attached to the carrier by slowly stirring, and if it is left to stand, the suspension culture can be continuously implemented again; (3) The differentiation ability and migration ability of stem cells (such as mesenchymal stem cells) proliferated by using the liquid culture medium are maintained at a very high level; (4) When using the liquid culture medium, the cultured cells can be maintained for a long time under low serum conditions, and cell secretions can be efficiently produced; (5) When using the liquid culture medium, the dispersibility of spheres can be maintained for a long time; etc., and based on the above-mentioned insights, the present invention was completed by continuing research. That is, the present invention is as follows.

[0012] [1] A medium composition for suspension culture of adherent cells, comprising:

[0013] (1) Chitin nanofibers; and

[0014] (2) Chitosan nanofibers or polysaccharides.

[0015] [2] The medium composition for suspension culture according to [1], wherein the medium composition comprises chitin nanofibers and chitosan nanofibers, and the ratio of chitin nanofibers to chitosan nanofibers is chitin nanofibers:chitosan nanofibers=1:0.5-20.

[0016] [3] The medium composition for suspension culture according to [1], wherein the medium composition comprises chitin nanofibers and a polysaccharide, and the polysaccharide is selected from the group consisting of methylcellulose, deacylated gellan gum, and sodium alginate.

[0017] [4] The medium composition for suspension culture according to any one of [1] to [3], wherein the adherent cells are adherent cells that self-aggregate under suspension culture.

[0018] [5] The medium composition for suspension culture according to any one of [1] to [4], wherein the adhesive cells are stem cells.

[0019] [6] The medium composition for suspension culture according to [5], wherein the stem cells are mesenchymal stem cells.

[0020] [7] The medium composition for suspension culture according to [5] or [6], wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of stem cells.

[0021] [8] A method for culturing adherent cells, comprising the step of suspending and culturing the adherent cells in the following culture medium composition, wherein the culture medium composition comprises:

[0022] (1) Chitin nanofibers; and

[0023] (2) Chitosan nanofibers or polysaccharides.

[0024] [9] The culture method as described in [8], wherein the culture medium composition contains chitin nanofibers and chitosan nanofibers, and the ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers = 1:0.5-20.

[0025]

[10] The culture method according to [8], wherein the culture medium composition comprises chitin nanofibers and a polysaccharide, and the polysaccharide is selected from the group consisting of methylcellulose, deacylated gellan gum, and sodium alginate.

[0026]

[11] The culture method according to any one of [8] to

[10] , wherein the adherent cells are adherent cells that self-aggregate under suspension culture.

[0027]

[12] The culture method according to any one of [8] to

[11] , wherein the adhesive cells are stem cells.

[0028]

[13] The culture method according to

[12] , wherein the stem cells are mesenchymal stem cells.

[0029]

[14] The culture method according to

[12] or

[13] , wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of stem cells.

[0030]

[15] The culture method according to any one of [8] to

[14] , further comprising the following steps:

[0031] Step (1) does not perform a treatment to remove the suspension-cultured cells from the chitin nanofibers and chitosan nanofibers or polysaccharides, and further adds the suspension culture medium composition described in any one of [1] to [7]; and

[0032] (2) The mixture obtained in step (1) is subjected to suspension culture.

[0033]

[16] A method for producing cell secretions, comprising the step of culturing adherent cells in suspension in the following medium composition, wherein the medium composition comprises:

[0034] (1) Chitin nanofibers; and

[0035] (2) Chitosan nanofibers or polysaccharides.

[0036]

[17] The method as described in

[16] , wherein the culture medium composition contains chitin nanofibers and chitosan nanofibers, and the ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers = 1:0.5-20.

[0037]

[18] The method of

[16] , wherein the culture medium composition comprises chitin nanofibers and a polysaccharide, and the polysaccharide is selected from the group consisting of methylcellulose, deacylated gellan gum, and sodium alginate.

[0038]

[19] The method according to any one of

[16] to

[18] , wherein the adherent cells are adherent cells that autoaggregate under suspension culture.

[0039]

[20] The method according to any one of

[16] to

[19] , wherein the adhesive cells are stem cells.

[0040]

[21] The method of

[20] , wherein the stem cells are mesenchymal stem cells.

[0041]

[22] The method according to any one of

[16] to

[21] , wherein the concentration of serum in the culture medium composition is 2% or less.

[0042]

[23] The method according to any one of

[16] to

[22] , wherein the cell secretion is at least one selected from the group consisting of low molecular weight compounds, proteins, nucleic acids, and cell secretory vesicles.

[0043]

[24] A medium composition for culturing adhesive cells in suspension, comprising poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation, wherein the specific degree of acetylation is 5 to 70%.

[0044]

[25] The medium composition for suspension culture as described in

[24] , wherein the concentration of poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation in the medium composition is 0.0001 to 0.2% (w / v).

[0045]

[26] The medium composition for suspension culture according to

[24] or

[25] , wherein the adherent cells are adherent cells that self-aggregate under suspension culture.

[0046]

[27] The medium composition for suspension culture according to any one of

[24] to

[26] , wherein the adhesive cells are stem cells.

[0047]

[28] The medium composition for suspension culture according to

[27] , wherein the stem cells are mesenchymal stem cells.

[0048]

[29] The medium composition for suspension culture according to

[27] or

[28] , wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of stem cells.

[0049]

[30] A method for culturing adhesive cells, comprising the step of suspending and culturing the adhesive cells in a culture medium composition comprising poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation, wherein the specific degree of acetylation is 5 to 70%.

[0050]

[31] The culture method according to

[30] , wherein the concentration of poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation in the culture medium composition is 0.0001 to 0.2% (w / v).

[0051]

[32] The culture method according to

[30] or

[31] , wherein the adherent cells are adherent cells that self-aggregate under suspension culture.

[0052]

[33] The culture method according to any one of

[30] to

[32] , wherein the adhesive cells are stem cells.

[0053]

[34] The culture method according to

[33] , wherein the stem cells are mesenchymal stem cells.

[0054]

[35] The culture method according to

[33] or

[34] , wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of stem cells.

[0055]

[36] The culture method according to any one of

[30] to

[35] , further comprising the following steps:

[0056] In step (1), the suspension culture medium composition described in any one of

[24] to

[29] is further added without performing a treatment to peel the suspension cultured cells from the poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation; and

[0057] Step (2) The mixture obtained in step (1) is subjected to suspension culture.

[0058]

[37] A method for producing cell secretions, comprising the step of suspending and culturing adherent cells in a culture medium composition comprising poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation, wherein the specific degree of acetylation of the nanofibers is 5 to 70%.

[0059]

[38] The method as described in

[37] , wherein the concentration of poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation in the culture medium composition is 0.0001 to 0.2% (w / v).

[0060]

[39] The method according to

[37] or

[38] , wherein the adherent cells are adherent cells that autoaggregate under suspension culture.

[0061]

[40] The method according to any one of

[37] to

[39] , wherein the adhesive cells are stem cells.

[0062]

[41] The method of

[40] , wherein the stem cells are mesenchymal stem cells.

[0063]

[42] The method according to any one of

[37] to

[41] , wherein the concentration of serum in the culture medium composition is 2% or less.

[0064]

[43] The method according to any one of

[37] to

[42] , wherein the cell secretion is at least one selected from the group consisting of low molecular weight compounds, proteins, nucleic acids, and cell secretory vesicles.

[0065] In another embodiment, the present invention is as follows.

[0066] [1'] A medium composition for suspension culture of adherent cells, comprising chitin nanofibers and chitosan nanofibers.

[0067] [2'] The suspension culture medium composition according to [1'], wherein the ratio of chitin nanofibers to chitosan nanofibers is chitin nanofibers:chitosan nanofibers=1:0.5-20.

[0068] [3'] The medium composition for suspension culture according to [1'] or [2'], wherein the adhesive cells are stem cells.

[0069] [4'] The medium composition for suspension culture according to [3'], wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of the stem cells.

[0070] [5'] The medium composition for suspension culture according to [4'], wherein the stem cells are mesenchymal stem cells.

[0071] [6'] A method for culturing adherent cells, comprising the step of suspending and culturing the adherent cells in a culture medium composition comprising chitin nanofibers and chitosan nanofibers.

[0072] [7'] The culture method according to [6'], wherein the ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers=1:0.5-20.

[0073] [8'] The culture method according to [6'] or [7'], wherein the adhesive cells are stem cells.

[0074] [9'] The culture method according to [8'], wherein the suspension culture is used for proliferation of stem cells and for maintaining the pluripotency and migration of stem cells.

[0075] [10'] The culture method according to [9'], wherein the stem cells are mesenchymal stem cells.

[0076] [11'] The culture method according to any one of [6'] to [10'], further comprising the following steps:

[0077] Step (1) without performing a treatment to peel the suspension-cultured cells from the chitin nanofibers and chitosan nanofibers, and further adding the suspension culture medium composition described in any one of [1'] to [5']; and

[0078] Step (2) The mixture obtained in step (1) is subjected to suspension culture.

[0079] [12'] A method for producing cell secretions, comprising the step of suspending and culturing adherent cells in a culture medium composition comprising chitin nanofibers and chitosan nanofibers.

[0080] [13'] The method according to [12'], wherein the ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers = 1:0.5-20.

[0081] [14'] The method according to [12'] or [13'], wherein the concentration of serum in the culture medium composition is 2% or less.

[0082] [15'] The method according to any one of [12'] to [14'], wherein the adhesive cells are mesenchymal stem cells.

[0083] [16'] The method according to any one of [12'] to [15'], wherein the cell secretion product is at least one selected from the group consisting of low molecular weight compounds, proteins, nucleic acids, and cell secretory vesicles.

[0084] Effects of the Invention

[0085] According to the present invention, by culturing the adhesive cells in a state of adhering to nanofibers formed by water-insoluble polysaccharides, it is possible to suspend and culture in a static state without the need for operations such as oscillation and rotation that cause the risk of cell damage and loss of function. In addition, when the adhesive cells cultured by the present invention are stem cells, it is possible to efficiently prepare high-quality stem cells that maintain the characteristics of stem cells (for example, differentiation ability, migration and homing ability in mesenchymal stem cells, etc.) at a high level. In addition, according to the present invention, without using proteolytic enzymes such as trypsin to peel off the carrier from the cells adhered to the carrier, the expansion of the culture scale can be simply implemented only by slow stirring using blowing and suction and adding fresh culture medium. As a result, useful cell secretions can also be efficiently produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] [ Figure 1 ] Figure 1 This is a graph showing the number of viable cells over time when HEK293-IFNβ cells were suspended and cultured for 21 days using a culture medium composition to which sample 1 (chitin nanofibers), sample 6 (a mixture of chitin nanofibers and chitosan nanofibers), sample 14 (chitin / chitosan nanofibers prepared by simultaneous defibration), or sample 15 (nanofibers with an adjusted amount of N-acetylglucosamine (amount of N-acetylglucosamine: 50%)) was added. The four bar graphs for each sample represent the number of viable cells at the 0th day, the 7th day, the 14th day, and the 21st day, respectively, from the left.

[0087] [ Figure 2 ] Figure 2The photographs show the spheroid state of the cells (Day 8) when HEK293-IFNβ cells were suspension cultured using the medium composition to which each nanofiber was added.

[0088] [ Figure 3 ] Figure 3 The photographs show the spheroid state of the cells (day 15) when HEK293-IFNβ cells were suspension cultured using the medium composition to which each nanofiber was added.

[0089] [ Figure 4 ] Figure 4 The photographs show the spheroid state of the cells (day 21) when HEK293-IFNβ cells were suspension cultured using the medium composition to which each nanofiber was added. DETAILED DESCRIPTION

[0090] Hereinafter, the present invention will be described in detail.

[0091] 1. Medium composition for suspension culture of adherent cells

[0092] The present invention provides a medium composition for suspension culture of adherent cells comprising (1) chitin nanofibers; and (2) chitosan nanofibers or polysaccharides (hereinafter sometimes referred to as "the medium composition of the present invention"). The medium composition of the present invention contains chitin nanofibers and chitosan nanofibers or polysaccharides, so that adherent cells can be suspended and cultured without stirring, shaking, etc.

[0093] The adherent cells used in the present invention are cells that require a support such as a container wall for survival and growth.

[0094] In the present invention, the adhesive cells used are not particularly limited, and examples thereof include stem cells, progenitor cells, adult non-stem cells, primary cultured cells, cell lines, cancer cells, and the like. The so-called stem cells are cells that have both the ability to replicate themselves and the ability to differentiate into cells of other multiple systems. Examples of adhesive stem cells are not limited to the following, and adult stem cells such as mesenchymal stem cells, neural stem cells, hematopoietic stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, reproductive stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells can be cited. The so-called mesenchymal stem cells refer to stem cells that have the ability to differentiate into all or several of bone cells, chondrocytes, and adipocytes. Mesenchymal stem cells exist at low frequencies in tissues such as bone marrow, peripheral blood, umbilical cord blood, and adipose tissue, and can be separated from these tissues using known methods. The so-called progenitor cells refer to cells that are in the midway stage of differentiating from the above-mentioned stem cells into specific somatic cells and germ cells. Examples of adhesive progenitor cells include, but are not limited to, precursor adipocytes, precursor cardiomyocytes, precursor endothelial cells, neural progenitor cells, liver progenitor cells, pancreatic progenitor cells, kidney progenitor cells, etc. Examples of adhesive adult non-stem cells include, but are not limited to, fibroblasts, osteocytes, periosteal cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic β cells, melanocytes, etc. The so-called primary culture cells refer to cells in a state of culture before the cells and tissues separated from an organism are inoculated and the first passage is performed. Primary culture cells can be cells collected from any tissue such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain or nerve tissue. The so-called cell strain refers to a cell that has obtained unlimited proliferation ability through artificial manipulation in vitro. The adhesive cells used in the present invention are preferably stem cells or progenitor cells, more preferably mesenchymal stem cells.

[0095] The source of the adhesive cells used in the present invention is not particularly limited, and can be any cell from animals and plants. As animals, it is not limited, and examples thereof include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals, etc., preferably mammals. As examples of mammals, it is not limited, and rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cattle, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, macaques, chimpanzees, people, etc. can be mentioned. As plants, as long as the collected cells can be liquid cultured, it is not particularly limited. Examples include plants (e.g., medicinal ginseng, catharanthus roseus, scopolamine, coptis root, belladonna, etc.) that produce crude drugs (e.g., saponins, alkaloids, berberine, scopolamine, phytosterols, etc.); plants (e.g., blueberry, safflower, madder, saffron, etc.) that produce pigments and polysaccharides (e.g., anthocyanin, safflower pigment, madder pigment, crocin pigment, flavonoids, etc.) used as raw materials for cosmetics and foods, but are not limited thereto. In the present invention, it is preferred to use mammalian adhesive cells.

[0096] In one embodiment, the adhesive cells may be adhesive cells that self-aggregate under suspension culture. Adhesive cells that self-aggregate under suspension culture include cells that form spheres under suspension culture. As adhesive cells that self-aggregate under suspension culture, for example, somatic cells include HEK293 cells, HeLa cells, A549 cells, etc., and stem cells include mesenchymal stem cells, preadipocytes, iPS cells, etc. In one embodiment of the present invention, for adhesive cells that self-aggregate under suspension culture, HEK293 cells and HeLa cells are preferred as somatic cells, and mesenchymal stem cells and iPS cells are preferred as stem cells.

[0097] The chitin nanofibers and chitosan nanofibers or polysaccharides used in the present invention are dispersed in a liquid culture medium, and exhibit an effect of suspending the adhesive cells attached to the nanofibers or polysaccharides in the liquid culture medium.

[0098] The chitin nanofibers and chitosan nanofibers or polysaccharides used in the present invention have the following effects: when mixed with a liquid culture medium, the nanofibers are 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, and the cells are prevented from settling and adhering to the culture container. The so-called not substantially increasing the viscosity of the liquid means that the viscosity of the liquid does not exceed 8mPa·s. The viscosity of the liquid at this time (that is, the viscosity of the culture medium composition of the present invention described below) is 8mPa·s or less, preferably 4mPa·s or less, and more preferably 2mPa·s or less. The viscosity of the liquid containing chitin nanofibers and chitosan nanofibers or polysaccharides can be evaluated, for example, using a tuning fork vibration viscosity measurement (SV-1A, A&D Company Ltd.) at 25°C.

[0099] The chitin nanofibers and chitosan nanofibers used in the present invention are composed of chitin and chitosan (i.e., chitin-like substances) which are water-insoluble polysaccharides. The so-called sugars refer to sugar polymers formed by polymerization of more than 10 monosaccharides (e.g., trisaccharides, tetrasaccharides, pentoses, hexoses, heptoses, etc.).

[0100] The so-called chitin-like substance refers to one 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. Generally speaking, chitin-like substances with a high content of N-acetylglucosamine and poor solubility in acidic aqueous solutions are chitin, and chitosan-like substances with a high content of glucosamine and soluble in acidic aqueous solutions are chitosan. In this specification, for convenience, N-acetylglucosamine is sometimes called chitin when the proportion of the constituting sugar is 50% or more, and chitosan is called chitosan when it is less than 50%. In one embodiment, the proportion of N-acetylglucosamine in the sugar units constituting chitin can be 80% or more, 90% or more, 98% or more, or 100%. In addition, in one embodiment, the proportion of N-acetylglucosamine in the sugar units constituting chitosan can be less than 40%, less than 30%, less than 20%, or less than 10%.

[0101] As raw materials for chitin, various biological resources such as shrimp, crab, insect, shellfish, mushroom, etc. can be used. The chitin used in the present invention can be chitin with an α-type crystal structure such as chitin derived from crab shells and shrimp shells, or chitin with a β-type crystal structure such as chitin derived from cuttlebone. The shells of crabs and shrimps are often treated as industrial waste. From the perspective of easy availability and effective utilization, they are preferred as raw materials, but deproteinization and deashing processes are required to remove proteins, ash, etc. contained as impurities. Therefore, in the present invention, it is preferred to use purified chitin that has been subjected to a dematrix treatment. Purified chitin is already on sale. As the raw material for the chitin nanofiber used in the present invention, it can be chitin with any one of the α-type and β-type crystal structures, preferably α-type chitin.

[0102] Chitosan can be produced by deacetylation of chitin by boiling it in a concentrated alkali (eg, a concentrated NaOH aqueous solution). Commercially available chitosan can also be used.

[0103] By pulverizing the above-mentioned chitin and chitosan (sometimes also referred to as nanofibrillation or defibration), chitin nanofibers and chitosan nanofibers can be obtained. The pulverization method is not limited. In order to achieve a fiber diameter and fiber length that meet the purpose of the present invention, a method that can obtain a strong shear force, such as a medium stirring mill such as a high-pressure homogenizer, a grinder (stone mortar), or a bead mill, is preferred. The mixture of chitin nanofibers and chitosan nanofibers used in the present invention can be prepared by mixing the chitin nanofibers and chitosan nanofibers after defibration, respectively, or by mixing chitin powder with chitosan powder and defibration of the mixture at the same time.

[0104] Among them, it is preferred to use a high-pressure homogenizer for micronization, and it is desirable to use a wet pulverization method such as disclosed in Japanese Patent Publication No. 2005-270891 and Japanese Patent No. 5232976 for micronization (pulverization). Specifically, it can be implemented by using a device that pulverizes the raw material by spraying a dispersion obtained by dispersing the raw material from a pair of nozzles at high pressure and causing them to collide, such as Starburst System (a high-pressure pulverization device made by Sugino Machine Limited) and NanoVater (a high-pressure pulverization device made by Yoshida Machinery Industry (strain)).

[0105] When the raw material is finely divided (pulverized) using the above-mentioned high-pressure homogenizer, the degree of fineness and homogenization depends on the pressure of the ultra-high pressure chamber of the high-pressure homogenizer, the number of times the ultra-high pressure chamber is passed (the number of times of treatment), and the concentration of the raw material in the aqueous dispersion. The pressure of the pressure (treatment pressure) is not particularly limited, and is usually 50 to 250 MPa, preferably 100 to 200 MPa.

[0106] In addition, the concentration of the raw material in the aqueous dispersion during the micronization treatment is not particularly limited, and is generally 0.1% to 30% by mass, preferably 1% to 10% by mass. The number of micronization (pulverization) treatments is not particularly limited, and depends on the concentration of the raw material in the aqueous dispersion. When the concentration of the raw material is 0.1 to 1% by mass, the micronization can be sufficiently performed by about 10 to 100 times of treatment, but when the concentration is 1 to 10% by mass, there is a case where the number of treatments needs to be about 10 to 1000 times.

[0107] The viscosity of the aqueous dispersion during the above-mentioned micronization treatment is not particularly limited. For example, in the case of α-chitin, the viscosity of the aqueous dispersion is in the range of 1 to 100 mPa·S, preferably 1 to 85 mPa·S (measured by tuning fork vibration viscosity at 25°C (SV-1A, A&D Company Ltd.)). In addition, 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.)). In addition, the particle size of chitin or chitosan in the aqueous dispersion during the micronization treatment is not particularly limited. For example, in the case of α-chitin, the average particle size of α-chitin in the aqueous dispersion is in the range of 0.5 to 200 μm, preferably 30 to 150 μm (measured by laser diffraction / scattering particle size distribution measuring device LA-960 (Horiba Ltd.)). In the case of chitosan, the average particle size of chitosan in the aqueous dispersion is in the range of 0.5 to 300 μm, preferably 50 to 100 μm (using a laser diffraction / scattering particle size distribution analyzer LA-960 (Horiba Ltd.)).

[0108] The method for preparing nanofibers is described in WO2015 / 111686A1 and the like.

[0109] Polysaccharides may be added to the medium composition of the present invention. In the present specification, polysaccharides refer to sugar polymers formed by polymerization of 10 or more monosaccharides (e.g., trisaccharides, tetrasaccharides, pentoses, hexoses, heptoses, etc.).

[0110] Examples of the water-insoluble polysaccharides include celluloses such as cellulose and hemicellulose; and chitins such as chitin and chitosan. However, the water-insoluble polysaccharides are not limited thereto.

[0111] As water-soluble polysaccharides, acidic polysaccharides having anionic functional groups can be cited. Acidic polysaccharides having anionic functional groups are not particularly limited, and for example, polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid) in the structure; polysaccharides having sulfuric acid or phosphoric acid in the structure, or polysaccharides having these two structures, etc. can be cited. More specifically, examples include substances composed of one or more selected from hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, zanthan gum, hexuronic acid, fucoidan, pectin, pectinic acid, heparan sulfate, heparin, heparin sulfate, kerato sulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and salts thereof.

[0112] Examples of the salts include: salts of alkali metals such as lithium, sodium and potassium; salts of alkaline earth metals such as calcium, barium and magnesium; salts of aluminum, zinc, copper, iron and the like; ammonium salts; quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, hexadecyltrimethylammonium, benzylmethylhexyldecylammonium and choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diethanolamine, trishydroxymethylaminomethane, meglumine, procaine and chloroprocaine; salts with amino acids such as glycine, alanine and valine; and the like.

[0113] In one embodiment, as the polysaccharide, methylcellulose, deacylated gellan gum, alginic acid, xanthan gum, carrageenan, dithian gum, locust bean gum, tamarind gum, pectin, carboxymethylcellulose, or salts thereof can be preferably used.

[0114] In order to achieve the desired effect in the culture medium composition of the present invention, the mixing ratio of chitin nanofibers and chitosan nanofibers may be important. First, chitin nanofibers and chitosan nanofibers are blended in a ratio (weight) of chitin nanofibers: chitosan nanofibers = 1:0.5-20 (preferably chitin nanofibers: chitosan nanofibers = 1:0.5-10, more preferably chitin nanofibers: chitosan nanofibers = 1:0.7-9, further preferably chitin nanofibers: chitosan nanofibers = 1:1-8, further preferably chitin nanofibers: chitosan nanofibers = 1:2-7, particularly preferably chitin nanofibers: chitosan nanofibers = 1:3-6). The obtained chitin nanofiber / chitosan nanofiber mixture can be added to the liquid culture medium in such a manner that the concentration of the total nanofibers (chitin nanofibers and chitosan nanofibers) contained in the culture medium composition is generally 0.0001 to 0.2% (w / v), preferably 0.0005 to 0.1% (w / v), further preferably 0.001 to 0.05% (w / v), and particularly preferably 0.006 to 0.05% (w / v). Alternatively, the culture medium composition of the present invention can be prepared by adding the required amounts of chitin nanofibers and chitosan nanofibers to the liquid culture medium, respectively, and stirring sufficiently. In one embodiment, for the culture medium composition of the present invention, the following conditions are met regarding the concentration of the nanofibers:

[0115] (1) The concentration of the total nanofibers (chitin nanofibers and chitosan nanofibers) contained in the culture medium composition is 0.0001-0.2% (w / v), and the weight ratio of chitin nanofibers to chitosan nanofibers contained in the culture medium composition is 1:0.5-20 (preferably 1:0.5-10, 1:0.7-9, 1:1-8, 1:2-7, or 1:3-6);

[0116] (2) the concentration of the total nanofibers (chitin nanofibers and chitosan nanofibers) contained in the culture medium composition is 0.0005-0.1% (w / v), and the weight ratio of chitin nanofibers to chitosan nanofibers contained in the culture medium composition is 1:0.5-20 (preferably 1:0.5-10, 1:0.7-9, 1:1-8, 1:2-7, or 1:3-6);

[0117] (3) the concentration of the total nanofibers (chitin nanofibers and chitosan nanofibers) contained in the culture medium composition is 0.001 to 0.05% (w / v), and the weight ratio of chitin nanofibers to chitosan nanofibers contained in the culture medium composition is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1:1 to 8, 1:2 to 7, or 1:3 to 6); or

[0118] (4) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers) contained in the culture medium composition is 0.006-0.05% (w / v), and the weight ratio of chitin nanofibers:chitosan nanofibers contained in the culture medium composition is 1:0.5-20 (preferably 1:0.5-10, 1:0.7-9, 1:1-8, 1:2-7, or 1:3-6).

[0119] In addition, when chitin nanofibers and polysaccharides are used, the following concentrations can be exemplified, but are not limited to these:

[0120] Chitin nanofibers: methylcellulose = 0.0005-0.1% (w / v): 0.0004-0.4% (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.04% (w / v))

[0121] Chitin nanofibers: deacylated gellan gum = 0.0005-0.1% (w / v): 0.0001-0.1 (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.01% (w / v))

[0122] Chitin nanofibers: sodium alginate = 0.0005-0.1% (w / v): 0.0004-0.4% (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.04% (w / v))

[0123] Chitin nanofiber: tamarind gum = 0.0005-0.1% (w / v): 0.0004-0.4% (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.04% (w / v))

[0124] Chitin nanofibers: pectin = 0.0005-0.1% (w / v): 0.0004-0.4% (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.04% (w / v))

[0125] Chitin nanofibers: carboxymethyl cellulose = 0.0005-0.1% (w / v): 0.0004-0.4% (w / v) (more preferably 0.001-0.05% (w / v): 0.001-0.04% (w / v))

[0126] In addition, although not wishing to be limited by theory, it can be considered that the fact that the proportion of chitin nanofibers and chitosan nanofibers is important for obtaining the desired effect of the present invention suggests that the total amount of acetylated glucosamine in chitin nanofibers and chitosan nanofibers is important. Therefore, in one embodiment of the present invention, instead of using a mixture of two nanofibers, chitin nanofibers and chitosan nanofibers, nanofibers prepared from a single poly (1,4)-N-acetyl-β-D-glucosamine with an optimized degree of acetylation and a number of acetyl groups corresponding to the number of acetyl groups in the mixture of the two nanofibers (hereinafter sometimes referred to as "N-acetylglucosamine amount-adjusted nanofibers", etc.) can also be used. Therefore, in one embodiment of the present invention, a suspension culture medium for adherent cells containing poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation is also provided. The specific acetylation degree of the poly (1,4)-N-acetyl-β-D-glucosamine may be generally 5 to 70%, preferably 9 to 70%, more preferably 11 to 55%, further preferably 14 to 50%, further preferably 16 to 50%, and particularly preferably 22 to 33%, but is not limited to these as long as the desired effect can be obtained.

[0127] The poly (1,4)-N-acetyl-β-D-glucosamine having a specific degree of acetylation as described above can be produced by a known method. As an example, a method of deacetylation of chitin having a high acetylation ratio (e.g., 90% or more) by boiling in an alkali (e.g., a concentrated NaOH aqueous solution) to produce poly (1,4)-N-acetyl-β-D-glucosamine having a desired degree of acetylation can be cited, but the method is not limited thereto.

[0128] By micronizing the poly (1,4)-N-acetyl-β-D-glucosamine having a specific degree of acetylation thus obtained (sometimes also referred to as nanofibrillation or defibration) using the above-mentioned method, poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation can be prepared.

[0129] In one embodiment of the present invention, N-acetylglucosamine amount-adjusting nanofibers can be added to a liquid culture medium composition. As for the concentration of N-acetylglucosamine amount-adjusting nanofibers in a liquid culture medium composition, the nanofibers can be added to the liquid culture medium in such a manner that the concentration of the nanofibers contained in the culture medium composition becomes generally 0.0001 to 0.2% (w / v), preferably 0.0005 to 0.1% (w / v), further preferably 0.001 to 0.1% (w / v), and particularly preferably 0.006 to 0.06% (w / v), but the invention is not limited thereto as long as the desired effect can be obtained.

[0130] The culture medium contained in the culture medium composition of the present invention can be appropriately selected depending on the type of adhesive cells used, etc. For example, when the goal is to culture mammalian adhesive cells, a culture medium commonly used for culturing mammalian cells can be used as the culture medium contained in the culture medium composition of the present invention. Examples of the culture medium for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5A medium, Eagle MEM medium (EMEM), alpha MEM medium (alpha Modified Eagles's Minimum Essential Medium; αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Cambrex), and X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan H3000 (STEMCELL Technologies), StemSpanSFEM (STEMCELL Technologies), StemlineII (Sigma Aldrich), QBSF-60 (Qualitybiological), StemProhESCSFM (Invitrogen), mTeSR1 or mTeSR2 medium (STEMCELL Technologies), Sf-900II (Invitrogen), Opti-Pro (Invitrogen), etc.

[0131] Those skilled in the art can freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. in the above-mentioned culture medium according to the purpose. When cultivating mammalian cells, those skilled in the art can also add more than one other chemical components or biological components according to the purpose combination. As the components that can be added to the culture medium for mammalian cells, 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 matrixes, various cell adhesion molecules, etc. can be enumerated. Examples of cytokines that can be added to the culture 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 α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), etc., but are not limited to these.

[0132] Examples of hormones that can be added to the culture medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and 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 lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, and the like. The invention also includes, but is not limited to, leptin, luteinizing hormone, melanocyte stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcifediol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin and enkephalin.

[0133] Examples of growth factors that can be added to the culture 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 growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokines, Notch ligands (Delta1, etc.), Wnt proteins, angiopoietin-like proteins 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), pleiotrophin, and the like, but are not limited to these.

[0134] In addition, substances obtained by artificially changing the amino acid sequence of these cytokines and growth factors by genetic recombination technology can also be added. Examples thereof include IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0135] Examples of various extracellular matrices and various cell adhesion molecules include collagens I to XIX, fibronectin, laminins-1 to 12, Nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, agarose, hyaluronic acid, alginate gel, various hydrogels, and cleavage fragments thereof.

[0136] Examples of antibiotics that can be added to the culture medium include sulfonamides, penicillin, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclopentyl penicillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, mecillin, andinocillin, cephalosporins and their derivatives, oxolinic acid, amloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, mefloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulanic acid, β-bromopenicillanic acid, β-chloropenicillanic acid acid), 6-acetylmethylene-penicillanic acid, cefuroxime, sultamicillin, adiposhirin, sulbactam formaldehyde hudrate ester, tazobactam, aztreonam, sulfazethin, isosulfazethin, norcardicin, phenylacetamidophosphonicacid methyl, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

[0137] It should be noted that, as described above, serum and / or serum substitutes can be added to the culture medium composition of the present invention, and when the culture medium composition of the present invention is used, even if the serum and / or serum substitutes in the culture medium are set to a concentration lower than the concentration (e.g., 10% by weight) commonly used, the maintenance and / or expansion of cells can still be achieved. The concentration of the serum added to the culture medium composition of the present invention can be appropriately set according to the type of cells, culture conditions, culture purposes, etc., and as a mode, the concentration of the serum (and / or serum substitute) in the culture medium composition of the present invention can be set to less than 15% by weight, less than 10% by weight, less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, less than 0.9% by weight, less than 0.8% by weight, less than 0.7% by weight, less than 0.6% by weight, less than 0.5% by weight, less than 0.4% by weight, or less than 0.3% by weight. It should be noted that the concentration of serum can be set to a constant concentration during the culture period, and the concentration can also be increased or decreased as needed when the culture medium is replaced.

[0138] In one embodiment, when the purpose of cell culture is to produce cell secretions for regenerative medicine, etc., it is sometimes preferable to set the serum concentration in the medium composition of the present invention to a low level in order to reduce the serum risk. In this embodiment, the concentration of serum in the medium composition of the present invention can be set to, for example, 2% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, or 0.3% by weight or less.

[0139] In another embodiment, when maximizing the number of cells maintained and expanded in culture at a low serum concentration, the serum concentration can be set to generally 0.05 to 2.0 wt %, preferably 0.1 to 2.0 wt %, further preferably 0.2 to 2.0 wt %, and particularly preferably 0.2 to 1.0 wt %.

[0140] In another embodiment, the range of serum concentration for maximizing the production of cell secretions is generally 0.05 to 2.0 wt %, preferably 0.1 to 1.5 wt %, more preferably 0.2 to 0.9 wt %, and particularly preferably 0.4 to 1.0 wt %.

[0141] It should be noted that the time for maintaining and / or expanding the culture of cells in a low-serum medium can be appropriately set according to the type of cells, culture conditions, or culture purpose, and can be set, for example, to more than 1 day, more than 3 days, more than 5 days, more than 7 days, more than 9 days, more than 11 days, more than 13 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 40 days, more than 50 days, more than 60 days, more than 80 days, or more than 100 days.

[0142] In one embodiment, the culture time can be set to 1 to 200 days, 1 to 100 days, 1 to 80 days, 1 to 60 days, 1 to 50 days, 1 to 40 days, 1 to 30 days, 1 to 25 days, 1 to 20 days, 1 to 15 days, 1 to 13 days, 1 to 11 days, 1 to 9 days, 1 to 7 days, 1 to 5 days, or 1 to 3 days. In a more preferred embodiment, the culture time can be set to preferably 10 to 60 days, more preferably 15 to 50 days, further preferably 20 to 50 days, and particularly preferably 25 to 40 days.

[0143] The culture medium composition of the present invention can be prepared as follows: the chitin nanofibers and chitosan nanofibers or polysaccharides are mixed with a suitable liquid culture medium in a manner such that the adherent cells can be suspended (preferably suspended and allowed to stand) at a concentration that allows the adherent cells to be cultured in suspension using the culture medium composition.

[0144] In a preferred embodiment, the culture medium composition of the present invention can be prepared by mixing the dispersion in the aqueous solvent of the above-mentioned chitin nanofibers and chitosan nanofibers or polysaccharides with a liquid culture medium. The dispersion can be sterilized (autoclave, gamma ray sterilization, etc.). Alternatively, the dispersion can be sterilized for use after mixing with a liquid culture medium (aqueous solution of culture medium) prepared by dissolving a powder culture medium in water. The sterilization of the dispersion and the liquid culture medium can also be performed separately before mixing. As examples of aqueous solvents, water, dimethyl sulfoxide (DMSO) and the like can be cited, but are not limited to these. As an aqueous solvent, water is preferred. The aqueous solvent may contain appropriate buffers and salts. The dispersion of the above-mentioned nanofibers can be used as a culture medium additive for preparing the culture medium composition of the present invention.

[0145] The mixing ratio is not particularly limited, but the nanofiber dispersion:liquid culture medium (aqueous culture medium solution) (volume ratio) is usually 1:99 to 99:1, preferably 10:90 to 90:10, and more preferably 20:80 to 80:20.

[0146] In the present invention, the so-called suspension of cells refers to the state in which the cells are not adhered to the culture container (non-adhesion), and has nothing to do with whether the cells are precipitated. In addition, in the present invention, the following state is referred to as "suspended static": when the cells are cultured, the cells are dispersed in the liquid culture medium composition and are in a suspended state without external pressure, vibration, or oscillation, rotation operation, etc. on the liquid culture medium composition; the situation of culturing cells and / or tissues in this state is referred to as "suspended static culture". The time that can be suspended in "suspended static" is at least 5 minutes, preferably more than 1 hour, more than 24 hours, more than 48 hours, more than 6 days, more than 21 days, but as long as the suspended state is maintained, it is not limited to these times.

[0147] In a preferred embodiment, the medium composition of the present invention can achieve suspension and stillness of cells at at least one point in the temperature range (e.g., 0 to 40° C.) in which cells can be cultured. The medium composition of the present invention preferably achieves suspension and stillness of cells at at least one point in the temperature range of 25 to 37° C., most preferably at 37° C.

[0148] By culturing the adhesive cells under the state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides, the adhesive cells can be suspended and cultured. This suspension culture can be implemented by culturing the adhesive cells in the above-mentioned culture medium composition of the present invention. Chitin nanofibers and chitosan nanofibers or polysaccharides show the effect (preferably the effect of suspending and leaving them to stand) of the cells attached to the nanofibers or polysaccharides in the culture medium. In the culture medium composition of the present invention, chitin nanofibers and chitosan nanofibers or polysaccharides are dispersed when neither dissolving nor being attached to the culture vessel, and therefore, when culturing the adhesive cells in the culture medium composition, the adhesive cells are attached to the nanofibers or polysaccharides and are suspended in the culture medium composition. Based on this suspension effect, it is possible to increase the number of cells in each certain volume and cultivate compared to monolayer culture. In addition, in the suspension culture with rotation and oscillation operation in the past, the shear force for the cell is exerted, so there is a situation where the proliferation rate of the cell, the recovery rate is low or the function of the cell is impaired, but by using the culture medium composition of the present invention, the cell can be cultured in a dispersed state without the need for operations such as oscillation, so it is expected that the cell function is not lost, and the target adhesive cells are easily and in large quantities suspended. In addition, when suspending and culturing cells in the culture medium containing gel substrate in the past, there is a situation where the observation and recovery of the cell are difficult or its function is damaged during recovery, but by using the culture medium composition of the present invention, it is expected that the cells of the suspension culture are observed and recovered without damaging their function. In addition, there is a situation where the viscosity is high and the culture medium is difficult to replace in the culture medium containing gel substrate in the past, but the culture medium composition of the present invention is low in viscosity, so it is expected that the culture medium can be easily replaced using a pipette, a pump, etc.

[0149] In the case of using chitin nanofibers and chitosan nanofibers or polysaccharides to suspend and culture adhesive cells, in the culture medium composition of the present invention, add the adhesive cells prepared in addition, mix evenly. The mixing method at this time is not particularly limited, and can include, for example, the mixing manually carried out by blowing and suction, the mixing of the equipment such as magnetic agitator, vortex mixer (Vortex Mixer), microplate mixer, oscillator, etc. After mixing, the cell suspension obtained can be cultivated under static state, and it is also possible to cultivate while rotating, vibrating or stirring as required. Its rotating speed and frequency can be appropriately set according to the purpose of those skilled in the art. For example, adhesive cells are recovered from subculture, and suitable cell dissociation fluid is used to be dispersed to the state of single cells or close to single cells, and the dispersed adhesive cells are suspended in the culture medium composition of the present invention, and suspension culture (preferably suspension static culture) is carried out to it.

[0150] The temperature for culturing cells is usually 25 to 39° C., preferably 33 to 39° C. (e.g., 37° C.) for animal cells. The CO2 concentration is usually 4 to 10% by volume, preferably 4 to 6% by volume in the culture atmosphere. The culture time can be appropriately set according to the purpose of the culture.

[0151] The culture of the adhesive cell in the culture medium composition of the present invention can be implemented using culture vessels such as culture dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, pipes, dishes, culture bags, roller bottles that are generally used for the culture of cells. It is expected that these culture vessels are low-adhesion cells, so that the adhesive cells attached to nanofibers do not adhere to the culture vessels. As the culture vessel of low-adhesion cells, the surface of the culture vessel can be used without artificially processing (for example, using the coating treatment of extracellular matrix etc.) the purpose of the adhesion with cells based on the improvement, or the surface of the culture vessel has been artificially processed based on the purpose of reducing the adhesion with cells.

[0152] When it is necessary to replace the culture medium, after separating the cells by centrifugation or filtration, fresh culture medium or the culture medium composition of the present invention is added to the cells. Alternatively, after the cells are appropriately concentrated by centrifugation or filtration, fresh culture medium or the culture medium composition of the present invention is added to the concentrated solution. For example, the gravitational acceleration (G) during centrifugation is 100G to 400G, and the size of the micropores of the filter used during the filtration process is 10μm to 100μm, but it is not limited thereto.

[0153] The culture of adherent cells can be carried out using bioreactors and automatic culture devices that can automatically perform cell inoculation, culture medium replacement, cell image acquisition, culture cell recovery, control pH, temperature, oxygen concentration, etc. under mechanical control and in a closed environment, and can also achieve high-density culture.

[0154] When the adhesive cells are suspended and cultured under the state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides, since the adhesive cells can be efficiently proliferated, the suspension culture is excellent as a method for the proliferation of adhesive cells. When the adhesive cells are suspended and cultured under the state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides, the adhesive cells are not only locally present on the bottom surface of the culture vessel, but disperse in a three-dimensionally expanded manner, promoting proliferation. As a result, the cells of proliferation become the state connected to the nanofiber or polysaccharides in a grape string shape. For this proliferation promoting effect, as long as the nanofibers of sufficient concentration for making the adhesive cells suspend (that is, avoiding the adhesion of the adhesive cells to the culture vessel) are included in the culture medium composition, it is not necessary to realize suspension and stand still (that is, without pressure, vibration, oscillation, rotation operation, etc. from the outside, the cells are uniformly dispersed in the liquid culture medium composition and are in a suspended state).

[0155] When the adhesive cells are suspended and cultured in a state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides, and the cells are proliferated, the culture medium contained in the culture medium composition of the present invention used in the suspension culture can be a culture medium that can proliferate the cells while maintaining the properties of the adhesive cells. According to the type of the adhesive cells, those skilled in the art can appropriately select the culture medium.

[0156] In one embodiment, mammalian stem cells (e.g., mesenchymal stem cells) are suspended and cultured in a state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides to proliferate the cells. Through the suspension culture, the differentiation ability, homing and migration ability of mammalian stem cells (e.g., mesenchymal stem cells) can be maintained while proliferating. Therefore, when using the culture medium composition of the present invention, high-quality stem cells can be prepared. It should be noted that whether stem cells have maintained characteristics such as differentiation ability and migration ability can be distinguished by methods known to themselves. In short, it can be easily distinguished by determining the expression of cell markers (e.g., OCT4 gene, SOX2 gene, NANOG gene, etc.) related to undifferentiation in stem cells based on mRNA levels and / or protein levels, and cell markers (e.g., CXCR4 gene, etc.) related to migration.

[0157] In the case where the adhesive cells are suspended and cultured under the state of being attached to chitin nanofibers and chitosan nanofibers or polysaccharides, it is not necessary to peel off the operation of the cells from the culture vessel, only by adding fresh culture medium or culture medium composition of the present invention only to the suspension culture or adding all or part of the suspension culture to fresh culture medium or culture medium composition of the present invention, the adhesive cells can be subcultured. By using this subculture method, it is possible to subculture the adhesive cells without peeling off the operation of the cells from the culture vessel. In addition, by using this subculture method, it is possible to expand the culture scale of the adhesive cells without peeling off the operation of the cells from the culture vessel. As the operation of peeling off the cells from the culture vessel, the processing utilizing chelating agent (such as EDTA) and / or proteolytic enzyme (such as trypsin, collagenase) can be enumerated. The above-mentioned subculture method is conducive to the subculture of the adhesive cells (such as, the adhesive cells whose viability is reduced due to the peeling operation, the adhesive cells whose properties are easily changed due to the peeling operation) with high sensitivity to the operation of peeling off the cells from the culture vessel. Examples of adhesive cells that are highly sensitive to the operation of detaching the cells from the culture container include stem cells (eg, mesenchymal stem cells), progenitor cells (eg, preadipocytes), and primary cultured cells, but are not limited thereto.

[0158] Adhesive cells proliferated using the medium composition of the present invention can be recovered after chitin nanofibers and chitosan nanofibers are decomposed using one or more degrading enzymes such as chitinase, chitobiase, chitosanase, and β-1,3-glucanase to separate the nanofibers from the adhesive cells. It should be noted that Yatalase (TakaraBio Inc.) is a mixture comprising chitinase, chitobiase, chitosanase, and β-1,3-glucanase, and can be preferably used as a degrading enzyme for chitin and chitosan.

[0159] For example, chitin and chitosan degrading enzymes are added to a suspension of adhesive cells attached to chitin nanofibers and chitosan nanofibers, and the mixture is incubated for a sufficient time for the peeling of the adhesive cells. The incubation temperature of chitin and chitosan based on the degrading enzyme is generally 20° C. to 37° C. The incubation time also depends on the type of enzyme, etc., but is generally 5 to 60 minutes.

[0160] After the nanofibers are decomposed and the adherent cells are detached from the nanofibers, the detached adherent cells can be recovered by centrifuging the suspension.

[0161] Since damage to the adhesive cells thus recovered is minimized, the cells can be preferably used for functional analysis, transplantation, and the like.

[0162] 2. Culture Methods of Adherent Cells

[0163] The present invention also provides a method for culturing adherent cells (hereinafter sometimes referred to as "the culturing method of the present invention"), which comprises the step of suspending and culturing the adherent cells in a culture medium composition comprising (1) chitin nanofibers; and (2) chitosan nanofibers or polysaccharides.

[0164] The culture method of the present invention is characterized in that, uses the above-mentioned culture medium composition of the present invention to cultivate adhesive cells.Therefore, the culture medium composition, adhesive cells, culture conditions etc. in the culture method of the present invention are the same as those described with respect to the culture medium composition of the present invention.It should be noted that, as mentioned above, for the mode using chitin nanofiber and chitosan nanofiber, it is also possible to use N-acetylglucosamine amount to regulate nanofiber to replace these 2 kinds of nanofibers.

[0165] 3. Methods for producing cell secretions

[0166] The present invention also provides a method for producing cell secretions (hereinafter sometimes referred to as "the production method of the present invention"), which comprises the step of suspending and culturing adherent cells in a culture medium composition comprising (1) chitin nanofibers; and (2) chitosan nanofibers or polysaccharides. It should be noted that, as described above, for the method using chitin nanofibers and chitosan nanofibers, nanofibers with an adjusted amount of N-acetylglucosamine may be used instead of these two nanofibers.

[0167] The production method of the present invention is characterized in that the above-mentioned medium composition of the present invention is used to culture the adherent cells. Therefore, the medium composition, adherent cells, culture conditions, etc. in the culture method of the present invention are the same as those described for the medium composition of the present invention.

[0168] The adhesive cells used in the production method of the present invention are not particularly limited as long as they are cells that produce cell secretions. In one embodiment, the adhesive cells may be mesenchymal stem cells. It should be noted that the source of the mesenchymal stem cells may be any one of bone marrow, fat cells, umbilical cord, and dental pulp.

[0169] The cell secretions in the production method of the present invention may include any substances secreted by cells, such as low molecular weight compounds, proteins, nucleic acids (miRNA or mRNA, etc.), and cell secretory vesicles (see below).

[0170] Cell secretions that can be produced by the production method of the present invention include, for example, prostaglandin E2 (PGE2), basic fibroblast growth factor (bFGF), tumor necrosis factor-stimulating gene 6 (TSG-6), vascular endothelial growth factor (VEGF), indoleamine-2,3-dioxygenase (IDO), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), angiopoietin-1, etc., but are not limited to these.

[0171] In this specification, the so-called cell secretory vesicles, typically, refer to vesicles released from cells and having a size that can be confirmed using an electron microscope. The size of the cell secretory vesicles can be 1 to 1,000 nm, 10 to 500 nm, or 30 to 200 nm in terms of average particle size. Here, the average particle size refers to the average value of the diameter of each particle measured based on a dynamic light scattering method or using an electron microscope. The cell secretory vesicles can be composed of a lipid bilayer surrounding a biological molecule.

[0172] Specific examples of the cell secretory vesicles include, but are not limited to, membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (average particle size of 30 to 1,000 nm), exosome-like vesicles, exosomes (average particle size of 30 to 200 nm), ectosome-like vesicles, ectosomes, or exovesicles. In a preferred embodiment of the production method of the present invention, the cell secretory vesicles may be exosomes.

[0173] It should be noted that, in one mode of the production method of the present invention, a compound for starting and / or promoting the production of cell secretions may also be added. The compound for starting and / or promoting the production of cell secretions may be selected from known substances according to the cell secretions to be produced. The compound includes TNF-α, IFN-γ, and interleukin-1β (IL-1β), but is not limited to these. As an example, when the cell secretion is PGE2 or TSG-6, TNF-α may be preferably added to the culture medium composition. In addition, when the cell secretion is PGE2 or IDO, IFN-γ may be preferably added to the culture medium composition. It should be noted that the concentration of the compound for starting and / or promoting the production of cell secretions added to the culture medium composition is not particularly limited as long as the desired effect can be obtained, and it can be appropriately determined based on the combination of the cell secretion to be produced and the compound.

[0174] In addition, in another mode of the production method of the present invention, in order to increase the production of cell secretions, the cells and culture conditions used in the production method of the present invention can be appropriately optimized. As an example, there is a situation where adherent cells (such as mesenchymal stem cells) preferably exposed to hypoxic conditions are used in the manufacturing method of the present invention (see J Cell Mol Med. 2018 Mar; 22 (3): 1428-1442). The optimization of cells and / or culture conditions can use any technology known to itself.

[0175] The present invention is described in more detail in the following examples, but the present invention is not limited to these examples.

[0176] Example

[0177] (Test Example 1: Proliferation of human adipose tissue-derived mesenchymal stem cells in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers)

[0178] α-chitin nanofibers (ratio of N-acetylglucosamine: 95% or more) or chitosan nanofibers (ratio of N-acetylglucosamine: 20% or less) (biomass nanofiber BiNFi-S 2 mass%, Sugino Machine Limited) prepared based on the manufacturing method disclosed in WO2015 / 111686 were suspended in ultrapure water (Milli-Q water) in a manner of 1% (w / v), dispersed by inversion mixing, and the aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. The α-chitin nanofibers used were α-chitin nanofibers (sample 1) obtained by nanofiberization under the conditions of 200MPa and 5 passes. In addition, the chitosan nanofibers were chitosan nanofibers (sample 2) obtained by nanofiberization under the conditions of 200MPa and 5 passes. Then, sample 1 and sample 2 are mixed in a ratio (volume) of sample 1 to sample 2 of 50%:50% (sample 3), 33%:67% (sample 4), 25%:75% (sample 5), 20%:80% (sample 6), 15%:85% (sample 7), 10%:90% (sample 8), 5%:95% (sample 9), and 1%:99% (sample 10) to obtain a chitin nanofiber / chitosan nanofiber mixture (samples 3 to 10).

[0179] A medium composition in which samples 1 to 10 obtained above were added to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) as a serum medium at various final concentrations (final concentrations: 0.0006% (w / v), 0.002% (w / v), 0.006% (w / v), 0.02% (w / v)) and a medium composition (sample 11) not containing the above substrate were prepared. Next, cultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were suspended in each of the above-mentioned medium compositions in a manner to give 13333 cells / mL, and then inoculated at 150 μL / well in a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning, #3474). The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 10 days. 150 μL of ATP reagent (CellTiter-Glo TM The cells were suspended and allowed to stand at room temperature for about 10 minutes, and then the luminescence intensity (RLU value) was measured using FlexStation3 (Molecular Devices). The luminescence value of the culture medium itself was subtracted to calculate the number of viable cells (average value of 3 points).

[0180] As a result, when mesenchymal stem cells derived from human fat were cultured in a 96-well flat-bottom ultra-low adhesion surface microplate using a culture medium composition comprising a mixture of α-chitin nanofibers and chitosan nanofibers, the total amount of α-chitin nanofibers and chitosan nanofibers contained in the culture medium composition, when α-chitin nanofibers were 5% by weight or more, a cell proliferation promoting effect was shown. In addition, when the total amount of α-chitin nanofibers and chitosan nanofibers contained in the culture medium composition, when α-chitin nanofibers were 25% by weight or more, a cell proliferation promoting effect of the same level was shown compared to the case of adding α-chitin nanofibers alone. The RLU values ​​(ATP determination, luminescence intensity) in each culture are shown in Tables 1, 2, and 3.

[0181] [Table 1]

[0182]

[0183] [Table 2]

[0184]

[0185] [Table 3]

[0186]

[0187] (Test Example 2: Proliferation of human bone marrow tissue-derived mesenchymal stem cells in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers)

[0188] After α-chitin nanofibers or chitosan nanofibers (BiNFi-S 2 mass%, Sugino Machine Limited) prepared based on WO2015 / 111686 were suspended in ultrapure water (Milli-Q water) in a manner of 1% (w / v), the aqueous solution was sterilized in an autoclave at 121°C for 20 minutes by inversion mixing dispersion. The α-chitin nanofibers used were α-chitin nanofibers (sample 1) obtained by nanofiberization under the conditions of 200MPa and 5 passes. In addition, the chitosan nanofibers were chitosan nanofibers (sample 2) obtained by nanofiberization under the conditions of 200MPa and 5 passes. Then, sample 1 and sample 2 were mixed in a ratio (volume) of 50%:50% (sample 3), 33%:67% (sample 4), and 20%:80% (sample 6) to obtain a chitin nanofiber / chitosan nanofiber mixture (samples 3, 4, and 6).

[0189] A medium composition in which samples 1 to 4 and 6 obtained above were added to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) as a serum medium at various final concentrations (final concentrations: 0.0006% (w / v), 0.002% (w / v), 0.006% (w / v), 0.02% (w / v)) and a medium composition (sample 11) not containing the above substrate were prepared. Next, cultured human bone marrow-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were suspended in each of the above-mentioned medium compositions in a manner to give 13333 cells / mL, and then inoculated at 150 μL / well in a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning, #3474). The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 10 days. Add 150 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (produced by Promega), suspended and allowed to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (produced by Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of live cells (average value of 3 points).

[0190] As a result, when mesenchymal stem cells derived from human bone marrow were cultured in a 96-well flat-bottom ultra-low adhesion surface microplate using a culture medium composition comprising a mixture of α-chitin nanofibers and chitosan nanofibers, when the total amount of α-chitin nanofibers and chitosan nanofibers contained in the culture medium composition was 20% by weight or more, the cell proliferation promoting effect was shown to be the same as that of the case of only α-chitin nanofibers (i.e., sample 1). The RLU values ​​(ATP determination, luminescence intensity) in each culture are shown in Table 4.

[0191] [Table 4]

[0192]

[0193] (Test Example 3: Continuous Expansion of Human Adipose Tissue-Derived Mesenchymal Stem Cells in 3D Culture Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0194] After α-chitin nanofibers or chitosan nanofibers (BiNFi-S 2 mass%, Sugino Machine Limited) prepared based on WO2015 / 111686 were suspended in ultrapure water (Milli-Q water) in a manner of 1% (w / v), the aqueous solution was sterilized in an autoclave at 121°C for 20 minutes by inversion mixing dispersion. The α-chitin nanofibers (sample 1) obtained by nanofiberization under 200MPa and 5 passes, and the chitosan nanofibers (sample 2) obtained by nanofiberization under 200MPa and 5 passes were used for mixing. Then, sample 1 and sample 2 were mixed in a ratio (volume) of 50%:50% (sample 3), 25%:75% (sample 4), 20%:80% (sample 6), and 15%:85% (sample 7) to obtain a chitin nanofiber / chitosan nanofiber mixture (samples 3, 4, 6, and 7).

[0195] A medium composition was prepared in which samples 1 to 4, 6, and 7 obtained above were added to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) as a serum medium at a final concentration of 0.006% (w / v). It should be noted that the final concentration of chitin nanofibers and / or chitosan nanofibers in the prepared medium composition is as follows:

[0196] (1) Culture medium composition to which sample 1 was added (chitin nanofiber: 0.006% (w / v), chitosan nanofiber: 0% (w / v))

[0197] (2) Culture medium composition to which sample 2 was added (chitin nanofiber: 0% (w / v), chitosan nanofiber: 0.006% (w / v))

[0198] (3) Culture medium composition to which sample 3 was added (chitin nanofiber: 0.003% (w / v), chitosan nanofiber: 0.003% (w / v))

[0199] (4) Culture medium composition to which sample 4 was added (chitin nanofiber: 0.0015% (w / v), chitosan nanofiber: 0.0045% (w / v))

[0200] (5) Culture medium composition to which sample 6 was added (chitin nanofiber: 0.0012% (w / v), chitosan nanofiber: 0.0048% (w / v))

[0201] (6) Culture medium composition to which sample 7 was added (chitin nanofiber: 0.0009% (w / v), chitosan nanofiber: 0.0051% (w / v))

[0202] Next, cultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were suspended in each of the above-mentioned culture medium compositions at a density of 13,333 cells / mL and then inoculated at 150 μL / well in a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning, #3474). The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 5 days. 150 μL of ATP reagent (CellTiter-Glo) was added to the culture medium at the time of inoculation (day 0) and on the 5th day after inoculation. TM The cells were suspended and allowed to stand at room temperature for about 10 minutes. The luminescence intensity (RLU value) was measured using FlexStation3 (Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of live cells (average value of 3 points).

[0203] On the 5th day after inoculation, the nanofibers to which the cells were attached were dispersed by pipetting, and the suspension was recovered from the 96-well flat-bottom ultra-low adhesion surface microplate, mixed with 600 μL of mesenchymal stem cell proliferation medium containing new samples (0.006%) by pipetting, and inoculated in a 24-well flat-bottom ultra-low adhesion surface microplate (made by Corning, #3473) with a total amount / well (24-well microplate inoculation day 0). The cell culture fluid on the 5th and 10th days after inoculation of the 24-well microplate was recovered into 1.5 mL tubes, respectively, and centrifuged at 2800 g for 3 minutes to allow the cell·nanofiber part to settle. 150 μL of mesenchymal stem cell proliferation medium and 150 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (produced by Promega), suspended and allowed to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (produced by Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of live cells (average value of 3 points).

[0204] On the 10th day after inoculation in the 24-well microplate, the nanofibers to which the cells were attached were dispersed by pipetting, and the suspension was recovered from the 24-well flat-bottom ultra-low adhesion surface microplate, mixed with 3000 μL of mesenchymal stem cell proliferation medium containing new samples (0.006%) by pipetting, and inoculated in a 6-well flat-bottom ultra-low adhesion surface microplate (made by Corning, #3471) with a total amount / well (6-well microplate inoculation day 0).

[0205] The cell culture medium on the 8th day after inoculation in the 6-well microplate was suspended by pipetting, and the mesenchymal stem cell proliferation medium was added to make the total volume 3500 μL, taking into account the evaporated portion. 500 μL of cell culture medium and 500 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay, Promega), suspended, left to stand at room temperature for about 10 minutes, FlexStation3 (Molecular Devices) was used to measure the luminescence intensity (RLU value), subtract the luminescence value of the culture medium itself, and calculate the number of living cells (average of 3 points). In addition, the final ATP value was converted in a way that the total amount became 3500 μL.

[0206] As a result, when using a culture medium composition containing α-chitin nanofibers and chitosan nanofibers in which the total amount of α-chitin nanofibers and chitosan nanofibers contained in the culture medium composition is 15% by weight to 50% by weight, and when culturing mesenchymal stem cells derived from human fat, it is possible to repeat the gentle aggregation between the fibers and the dispersion by blowing and suction. In addition, by adding a culture medium composition containing fresh α-chitin nanofibers and chitosan nanofibers, it is possible to achieve expanded culture without the use of trypsin. In particular, when the total amount of α-chitin nanofibers and chitosan nanofibers contained in the culture medium composition is 15% by weight to 20% by weight, when continuously expanding the culture, it is not easy to cause the cell proliferation rate to decrease due to the aggregation of mesenchymal stem cells derived from human fat on the nanofibers, and even in a 6-well plate, a good proliferation promotion effect is shown. The RLU values ​​(ATP determination, luminescence intensity) in each culture are shown in Table 5.

[0207] [Table 5]

[0208]

[0209] (Test Example 4: Changes in stem cell marker expression of human adipose-derived mesenchymal stem cells in 3D culture using chitosan nanofibers or a mixture of chitin nanofibers and chitosan nanofibers)

[0210] The α-chitin nanofiber or chitosan nanofiber (BiNFi-S 2 mass%, Sugino Machine Limited) prepared based on WO2015 / 111686 was suspended in ultrapure water (Milli-Q water) in a manner of 1% (w / v), and then dispersed by inversion mixing, and the aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. The α-chitin nanofiber used was an α-chitin nanofiber (sample 1) obtained by nanofiberization under the conditions of 200MPa and 5 passes. In addition, the chitosan nanofiber was a chitosan nanofiber (sample 2) obtained by nanofiberization under the conditions of 200MPa and 5 passes. Then, the sample 1 and the sample 2 were mixed in a manner such that the ratio (volume) of the sample 1 to the sample 2 was 50%:50% (sample 3), 33%:67% (sample 4), and 25%:75% (sample 5), and a chitin nanofiber / chitosan nanofiber mixture (samples 3 to 5) was obtained. A medium composition in which samples 1 to 5 obtained above were added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by TakaraBio Inc.) at a final concentration of 0.015% (w / v) and a medium composition (sample 11) not containing the above substrate were prepared. It should be noted that the final concentration of chitin nanofibers and / or chitosan nanofibers in the prepared medium composition is as follows:

[0211] (1) Culture medium composition to which sample 1 was added (chitin nanofiber: 0.015% (w / v), chitosan nanofiber: 0% (w / v))

[0212] (2) Culture medium composition to which sample 2 was added (chitin nanofiber: 0% (w / v), chitosan nanofiber: 0.015% (w / v))

[0213] (3) Culture medium composition to which sample 3 was added (chitin nanofiber: 0.0075% (w / v), chitosan nanofiber: 0.0075% (w / v))

[0214] (4) Culture medium composition to which sample 4 was added (chitin nanofiber: 0.005% (w / v), chitosan nanofiber: 0.01% (w / v))

[0215] (5) Culture medium composition to which sample 5 was added (chitin nanofiber: 0.00375% (w / v), chitosan nanofiber: 0.01125% (w / v))

[0216] Next, the cultured mesenchymal stem cells derived from human fat (C-12977, manufactured by Takara Bio Inc.) were inoculated in the above-mentioned culture medium composition with chitin and / or chitosan nanofibers or without the addition of the culture medium composition in a manner of 50,000 cells / mL, and then dispensed into the wells of a 6-well flat-bottom ultra-low adhesion surface microplate (#3471, manufactured by Corning) in a manner of 2 mL per well. In addition, as a comparison object, 25,000 cells / mL were inoculated in the wells of a 6-well flat-bottom adhesion surface microplate (#3516, manufactured by Corning) in a manner of 2 mL per well (sample 12). Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 6 days. On the 6th day, the cells were collected and 350 μL (RNeasy mini kit (manufactured by QIAGEN, #74106) RLT solution was added to prepare an RNA extraction solution. After adding 350 μL of 70% ethanol to the RNA extraction solution, it was added to the RNeasy purification column and centrifuged at 8000×g for 15 seconds. Next, 700 μL of RW1 solution was added to the RNeasy purification column and centrifuged at 8000×g for 15 seconds. Next, 500 μL of RPE solution was added and centrifuged at 8000×g for 15 seconds. Further 500 μL of RPE solution was added and centrifuged at 8000×g for 2 minutes. RNase-free solution was added to the RNA present in the RNeasy purification column to elute it. Then, cDNA was synthesized from the obtained RNA using PrimeScriptRT reagent Kit (Perfect Real Time) (manufactured by Takara Bio Inc., #RR037A). The synthesized cDNA and Premix EX Taq (Perfect Real Time) (Takara Bio Inc., #RR039A), Taq man probe (Applied Bio Systems) were used to perform real-time PCR. As Taq man probe (Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs01053049_s1 was used for SOX2, Hs04399610_g1 was used for NANOG, Hs00607978s1 was used for CXCR4, and Hs99999905_m1 was used for GAPDH. RealTime PCR7500 was used as the instrument. For analysis, the relative values ​​of the values ​​of each target gene corrected based on the values ​​of GAPDH were calculated and compared.

[0217] As a result, by using a culture medium composition comprising a mixture of chitosan nanofibers and α-chitin nanofibers and chitosan nanofibers, mesenchymal stem cells derived from human fat were cultured in a 6-well flat-bottom ultra-low adhesion surface microplate, and the relative expression of the OCT4, SOX2, NANOG genes reflecting undifferentiation and the CXCR4 gene reflecting the migration associated with homing was confirmed to rise. On the other hand, under the conditions of monolayer culture in an adhesion plate and 3D culture in a low adhesion plate using only chitin nanofibers, no significant increase in the expression of these genes was confirmed. The relative values ​​of each gene expression are shown in Table 6.

[0218] [Table 6]

[0219]

[0220] It should be noted that the physical properties (measured concentration of α-chitin or chitosan, viscosity, median particle size, average particle size, etc.) of the α-chitin nanofiber dispersion and chitosan nanofiber dispersion prepared under various defibration conditions (pressure·number of shredding) used in Test Examples 1 to 4 (and Test Examples 5, 6, and 7 described later) are shown in Table 7.

[0221] [Table 7]

[0222]

[0223] (Test Example 5: Continuous Expansion Culture of Human Umbilical Cord-Derived Mesenchymal Stem Cells in 3D Culture Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0224] A medium composition was prepared in which the sample 6 obtained above was added to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) as a serum medium in a final concentration of 0.05% (w / v) (hereinafter, the medium composition prepared herein is sometimes referred to as "mesenchymal stem cell proliferation medium containing nanofibers"). It should be noted that the final concentrations of chitin nanofibers and chitosan nanofibers in the prepared mesenchymal stem cell proliferation medium containing nanofibers are as follows: chitin nanofibers: 0.01% (w / v), chitosan nanofibers: 0.04% (w / v).

[0225] Next, cultured human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) were suspended in the above-mentioned mesenchymal stem cell proliferation medium containing nanofibers at a density of 15,000 cells / mL, and then inoculated into a 6-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning, #3471) at 10 mL / well. The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. During culture, the cells adhered to the nanofibers precipitated at the bottom of the culture wells, but did not adhere to the surface of the culture wells. On the 3rd day, about 5 mL of the culture supernatant in the wells that did not contain substantially any nanofiber / cell precipitates was removed, 5 mL of fresh mesenchymal stem cell proliferation medium containing nanofibers was added, the cells were suspended with a pipette, and culture was continued until the 7th day. 300 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (produced by Promega), suspended and allowed to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (produced by Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of live cells (average of 2 points).

[0226] On the 7th day after inoculation, the nanofibers to which the cells adhered were dispersed by pipetting, and the suspension was recovered from a 6-well flat-bottom ultra-low adhesion surface microplate, mixed with fresh mesenchymal stem cell proliferation medium (40 mL) containing nanofibers by pipetting, and inoculated into a 125 mL flask (manufactured by Thermo Scientific, 4115-0125) with a total amount / flask for culture. On the 10th day, about 25 mL of the culture medium in the flask was removed, and 25 mL of fresh mesenchymal stem cell proliferation medium containing nanofibers was added, and the cells were suspended with a pipette and cultured continuously until the 14th day after inoculation. 500 μL of ATP reagent (CellTiter-Glo) was added to 500 μL of the cell culture medium on the 7th day after expansion in the 125 mL flask. TM Luminescence cell viability assay, Promega), suspended, left to stand at room temperature for about 10 minutes, FlexStation3 (Molecular Devices) was used to measure the luminescence intensity (RLU value), subtracted the luminescence value of the culture medium itself, and the number of living cells (average of 2 points) was calculated. In addition, the final ATP value in the flask was converted using the expansion ratio (5 times) from 6 wells to the flask.

[0227] As a result, when using the culture medium composition containing α-chitin nanofibers and chitosan nanofibers to cultivate the mesenchymal stem cells derived from human umbilical cord, the agglutination and dispersion of the fibers can be repeated. In addition, by adding the culture medium composition containing fresh α-chitin nanofibers and chitosan nanofibers, it is possible to achieve expanded cultivation without using trypsin. The RLU values ​​(ATP determination, luminous intensity) in each culture are shown in Table 8.

[0228] [Table 8]

[0229]

[0230] (Test Example 6: Suspension culture of human umbilical cord-derived mesenchymal stem cells at low serum concentration using a culture medium composition containing chitin nanofibers and chitosan nanofibers, and continuous collection of cell secretions 1)

[0231] The 125 mL flask culture medium of the 14th day of culturing mesenchymal stem cells derived from human umbilical cord using the culture medium composition containing α-chitin nanofibers and chitosan nanofibers (i.e., a mesenchymal stem cell proliferation medium containing nanofibers) in Test Example 5 was used. On the 14th day, about 25 mL of the culture medium supernatant containing substantially no nanofiber / cell precipitate in the flask was removed, 25 mL of fresh DMEM culture medium (044-29765, FUJIFILM Wako Pure Chemical Corporation, containing no nanofibers and serum) was added, and the culture was suspended by a pipette, and the culture was continued to the 17th day. Then, on the 17th day, about 25 mL of the culture medium supernatant containing substantially no nanofiber / cell precipitate in the flask was recovered, 25 mL of fresh DMEM culture medium (044-29765, FUJIFILM Wako Pure Chemical Corporation, containing no nanofibers and serum) was added to the well, and the culture was suspended by a pipette, and the culture was continued to the 21st day. Then, on days 24, 28, 31, and 35, about 25 mL of the culture medium supernatant in the flask was recovered, and 25 mL of fresh DMEM medium: mesenchymal stem cell proliferation medium (3:1) (neither medium contained nanofibers) was added to the wells, suspended using a pipette, and cultured until day 35.

[0232] On the 21st, 24th, 28th, 31st, and 35th days, the recovered culture supernatant was centrifuged (400g, 3 minutes) and the culture supernatant was recovered. The recovered culture supernatant was stored at -80°C until use. In addition, the precipitate (nanofibers / cells, etc.) after centrifugation was returned to the flask in the cell culture and the culture in the flask was continued. That is, in all the above steps, the nanofiber concentration in the culture medium was always constant.

[0233] On days 14, 21, 28, and 35, 500 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (produced by Promega), suspended and allowed to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (produced by Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of live cells (average of 2 points).

[0234] [Measurement of exosome production using the enzyme-labeled antibody method]

[0235] The amount of exosomes produced in each culture supernatant (days 17, 21, 24, 28, 31, and 35) was measured using an enzyme-linked immunosorbent assay (ELISA). TM Exosome ELISA Kit (Wako Pure Chemical Industries, Ltd., #297-79201). Repeat the operation of adding reaction / washing solution to the ExosomeCapture 96Well Plate at 300 μL / well three times. Dispense the culture supernatant diluted 10 times at 100 μL / well, and react at room temperature for 2 hours using a microplate shaker. After the reaction is completed, discard the reaction solution, and repeat the operation of adding reaction / washing solution to each well at 300 μL / well three times. Dispense the reaction solution of the control anti-CD63 antibody for detection at 100 μL / well, and react at room temperature for 1 hour using a microplate shaker. After the reaction is completed, discard the reaction solution, and repeat the operation of adding reaction / washing solution to each well at 300 μL / well three times. Dispense the reaction solution of the secondary antibody for detection at 100 μL / well, and react at room temperature for 1 hour using a microplate shaker. After the reaction is completed, the reaction solution is discarded, and the operation of adding reaction / washing solution to each well at 300 μL / well is repeated 5 times. TMB solution is dispensed at 100 μL / well and allowed to react at room temperature for 30 minutes. After the reaction, stop solution is added at 100 μL / well, and the absorbance at 450 nm and 620 nm is measured. The absorbance value of each sample is set to the value obtained by subtracting the absorbance at 620 nm from the absorbance at 450 nm (ΔAbs).

[0236] As a result, mesenchymal stem cells derived from human umbilical cords proliferated on a medium composition containing α-chitin nanofibers and chitosan nanofibers still maintained the number of viable cells to the 35th day in a low serum medium with a ratio of 25% of the mesenchymal stem cell medium. In addition, for the CD63 value (exosome) in each culture supernatant, the production volume at 25% was higher than that at a ratio of 50% of the mesenchymal stem cell medium. In addition, the production volume increased with continued cultivation in a 25% low serum medium. The RLU values ​​(ATP determination, luminescence intensity) in each culture are shown in Table 9, and the ΔAbs values ​​from CD63 are shown in Table 10.

[0237] [Table 9]

[0238] Day 14 Day 21 Day 28 Day 35 Sample 6 14753 14934 16439 15320

[0239] [Table 10]

[0240]

[0241] (Test Example 7: Suspension culture of human umbilical cord-derived mesenchymal stem cells under stepwise low serum conditions using a culture medium composition containing chitin nanofibers and chitosan nanofibers, and recovery of cell secretions 2)

[0242] A medium composition was prepared in which samples 3 and 6 obtained above were added to a mesenchymal stem cell proliferation medium (C-28009, manufactured by Takara Bio Inc.) as a serum medium in a final concentration of 0.05% (w / v) (hereinafter, the medium composition prepared herein may be referred to as "mesenchymal stem cell proliferation medium containing nanofibers"). It should be noted that the final concentrations of chitin nanofibers and chitosan nanofibers in the prepared mesenchymal stem cell proliferation medium containing nanofibers are as follows:

[0243] (1) Culture medium composition to which sample 3 was added (chitin nanofiber: 0.025% (w / v), chitosan nanofiber: 0.025% (w / v))

[0244] (2) Culture medium composition to which sample 6 was added (chitin nanofiber: 0.01% (w / v), chitosan nanofiber: 0.04% (w / v))

[0245] Next, the cultured mesenchymal stem cells derived from human umbilical cord (C-12971, manufactured by Takara Bio Inc.) were suspended in the above-mentioned mesenchymal stem cell proliferation medium containing nanofibers in a manner of 15,000 cells / mL, and then inoculated into a 24-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning, #3473) at 1.2 mL / well. The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. During culture, the cells adhered to the nanofibers precipitated at the bottom of the culture wells, but no adhesion to the surface of the culture wells occurred. On the 3rd day, about 0.6 mL of the culture medium supernatant that did not substantially contain nanofiber / cell precipitates in the wells was removed, 0.6 mL of mesenchymal stem cell proliferation medium was added, suspended using a pipette, and cultured until the 6th day. On the 6th day after inoculation, the nanofibers to which the cells adhered were dispersed by pipetting, and the suspension was recovered from a 24-well flat-bottom ultra-low adhesion surface microplate, mixed with fresh mesenchymal stem cell proliferation medium (8.4 mL) containing nanofibers by pipetting, and inoculated into a 6-well flat-bottom ultra-low adhesion surface microplate (Corning, #3473) at 9.6 mL / well. The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 4 days. On the 10th day, about 5 mL of the culture medium supernatant in the well was removed, 5 mL of mesenchymal stem cell proliferation medium (without nanofibers) was added, suspended using a pipette, and cultured until the 14th day. On the 14th day after inoculation, the nanofibers to which the cells adhered were dispersed by pipetting, and the suspension was recovered from a 6-well flat-bottom ultra-low adhesion surface microplate, mixed with fresh mesenchymal stem cell proliferation medium (about 40 mL) containing nanofibers by pipetting, and inoculated into a 125 mL flask (Thermo Scientic, 4115-0125) with a total amount / flask for culture. On the 17th day, about 25 mL of the culture medium supernatant in the flask was removed, 25 mL of fresh mesenchymal stem cell proliferation medium (without nanofibers) was added, and the suspension was suspended by a pipette, and culture was continued until the 20th day after inoculation.

[0246] On the 20th day, about 25 mL of the supernatant of the culture medium in the flask was removed, and 25 mL of fresh DMEM culture medium (044-29765, FUJIFILM Wako Pure Chemical Corporation, without nanofibers and serum) was added, suspended by a pipette, and culture was continued until the 23rd day. Then on the 23rd day, about 25 mL of the supernatant of the culture medium in the flask was recovered, 25 mL of fresh DMEM culture medium (044-29765, FUJIFILM Wako Pure Chemical Corporation, without nanofibers and serum) was added to the well, suspended by a pipette, and culture was continued until the 26th day. Then on the 29th, 32nd, and 35th days, about 25 mL of the supernatant of the culture medium in the flask was recovered, 25 mL of fresh DMEM culture medium (without nanofibers and serum) was added to the well, suspended by a pipette, and culture was continued until the 35th day.

[0247] On the 26th, 29th, 32nd and 35th days, the recovered culture medium was centrifuged (400g, 3 minutes) and the culture supernatant was recovered. The recovered culture supernatant was stored at -80°C until use. In addition, the precipitate (nanofibers / cells, etc.) after centrifugation was returned to the flask in the cell culture and the culture in the flask was continued. That is, in all the above steps, the nanofiber concentration in the culture medium was always constant.

[0248] On days 26, 29, 32, and 35, 500 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (produced by Promega), suspended and allowed to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation3 (produced by Molecular Devices), and the luminescence value when only the culture medium was obtained was subtracted to calculate the number of live cells (average of 2 points).

[0249] [Measurement of exosome production using the enzyme-labeled antibody method]

[0250] The amount of exosomes produced in each culture supernatant (days 26, 29, 32, and 35) was measured using the enzyme-linked immunosorbent assay (ELISA). TMExosome ELISA Kit (Wako Pure Chemical Industries, Ltd., #297-79201). Repeat the operation of adding reaction / washing solution to the Exosome Capture 96Well Plate at 300 μL / well three times. Dispense the culture supernatant diluted 10 times at 100 μL / well, and use a microplate shaker to react at room temperature for 2 hours. After the reaction is completed, discard the reaction solution, and repeat the operation of adding reaction / washing solution to each well at 300 μL / well three times. Dispense the reaction solution of the control anti-CD63 antibody for detection at 100 μL / well, and use a microplate shaker to react at room temperature for 1 hour. After the reaction is completed, discard the reaction solution, and repeat the operation of adding reaction / washing solution to each well at 300 μL / well three times. Dispense the reaction solution of the secondary antibody for detection at 100 μL / well, and use a microplate shaker to react at room temperature for 1 hour. After the reaction is completed, the reaction solution is discarded and the operation of adding reaction / washing solution to each well at 300 μL / well is repeated 5 times. TMB solution is dispensed at 100 μL / well and allowed to react at room temperature for 30 minutes. After the reaction, stop solution is added at 100 μL / well and the absorbance at 450 nm and 620 nm is measured. The absorbance value of each sample is set to the value obtained by subtracting the absorbance at 620 nm from the absorbance at 450 nm (△Abs).

[0251] As a result, when DMEM medium was added to the mesenchymal stem cells derived from human umbilical cord proliferated on the medium composition containing α-chitin nanofibers and chitosan nanofibers in stages to make it a low serum medium, the ratio of the mesenchymal stem cell medium containing serum became 6.25%, and the number of viable cells decreased. On the other hand, with respect to the CD63 value (exosome) in each culture supernatant, the production volume decreased as the low serum level progressed, and the production volume was the highest under the condition that the ratio of the mesenchymal stem cell medium was 25% (i.e., the serum concentration was 0.5% by weight). The RLU values ​​(ATP determination, luminescence intensity) in each culture are shown in Table 11, and the ΔAbs values ​​from CD63 are shown in Table 12.

[0252] [Table 11]

[0253] Day 26 Day 29 Day 32 Day 35 Sample 3 894900 886450 568208 441348 Sample 6 527673 750692 410465 437438

[0254] [Table 12]

[0255]

[0256]

[0257] (Test Example 8: PGE2 Production in 3D Culture of Human Bone Marrow-Derived and Human Umbilical Cord-Derived Mesenchymal Stem Cells Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0258] A medium composition (final concentration: 0.05% (w / v)) (sample 6) in which chitin nanofibers and chitosan nanofibers are added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) and a medium composition (sample 12) not containing the above-mentioned substrate were prepared. Then, cultured human bone marrow-derived mesenchymal stem cells (C-12974, manufactured by Takara Bio Inc.) were inoculated at 40,000 cells / mL, and human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) were inoculated at 80,000 cells / mL in the above-mentioned medium composition to which chitin nanofibers and chitosan nanofibers were added or in which no medium composition was added, and then the medium composition to which chitin nanofibers and chitosan nanofibers were added was dispensed into the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning Incorporated) in a manner of 1 mL per well. In addition, as a comparison object, 1 mL or 2 mL was dispensed into the wells of a 24-well flat-bottom adhesive surface microplate (#3526, manufactured by Corning) or a 6-well flat-bottom adhesive surface microplate (#3516, manufactured by Corning) in a manner of 1 mL or 2 mL per well. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 2 days. On the second day, the culture medium composition with chitin nanofibers and chitosan nanofibers added and the culture medium composition not added were moved from each well to a 15 mL tube, centrifuged at 300 x g for 3 minutes, and the culture supernatant was recovered. After removing the culture supernatant, 1 mL of mesenchymal stem cell proliferation medium 2 medium or mesenchymal stem cell proliferation medium 2 medium containing TNF-α (#210-TA, manufactured by R&D System) at a final concentration of 10 ng / mL was added to a 15 mL tube, and the medium composition added with chitin nanofibers and chitosan nanofibers was returned to the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning). After removing the culture medium, 1 mL of mesenchymal stem cell proliferation medium 2 medium or mesenchymal stem cell proliferation medium 2 medium containing TNF-α at a final concentration of 10 ng / mL was added to the 24-well flat-bottom adhesion surface microplate (#3526, manufactured by Corning) was added. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2) for 1 day. On the first day, the culture medium composition to which chitin nanofibers and chitosan nanofibers were added and the culture medium composition without addition were transferred from each well to a 15 mL tube, and the culture supernatant was recovered after centrifugation at 300 xg for 3 minutes.At this time, in order to evaluate the number of cells, 1mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability detection, Promega company system) is added to each sample after the culture supernatant is recovered, and it is suspended, and after standing at room temperature for 10 minutes, Enspire (Perkin Elmer company system) is used to measure the luminescence intensity (RLU value), and the luminescence value of the culture medium itself is subtracted, and the number of living cells is thus measured. Then, about the PGE2 contained in the culture supernatant recovered, PGE2ELISA kit (#ADI-900-001, Enzo Life Sciences company system) is used for quantitative analysis. Standards (standard) and culture supernatants 100 μL diluted with assay buffer (Assay Buffer) are added to each well of the 96-well plate attached to the kit. Then, 50 μL of blue conjugates (blue conjugate) are added to each well. Then, 50 μL of yellow antibodies (yellowantibody) are added to each well, and they are shaken at room temperature for 2 hours. Next, discard the solution, add a wash solution at 400 μL / well, and discard the solution. Repeat the above operation 3 times. Add 200 μL of pNpp substrate solution to each well and shake at room temperature for 45 minutes. Finally, add 50 μL of stop solution to terminate the reaction and measure the absorbance at 405 nm. The PGE2 concentration contained in each sample is calculated by the four-parameter logistic regression of the standard curve. In order to calculate the secretion amount per unit cell number, the calculated PGE2 amount is divided by the luminescence intensity to calculate the relative value.

[0259] As a result, compared with the situation that monolayer culture is not added with culture medium composition and adhesion plate, the relative value of PGE2 secretion in the 3D culture of the culture medium composition added with chitin nanofiber and chitosan nanofiber and low adhesion plate combination increases. In addition, when comparing with regard to TNF-α, the culture medium composition added with chitin nanofiber and chitosan nanofiber, the 3D culture of low adhesion plate combination is still more than the situation that monolayer culture is not added with culture medium composition and adhesion plate. The relative value of each sample of the mesenchymal stem cell derived from human bone marrow and derived from human umbilical cord is shown in Table 13 and Table 14.

[0260] [Table 13]

[0261]

[0262] [Table 14]

[0263]

[0264] (Test Example 9: bFGF Production in 3D Culture of Human Bone Marrow- and Human Adipose-Derived Mesenchymal Stem Cells Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0265] A medium composition (final concentration: 0.05% (w / v)) in which chitin nanofibers and chitosan nanofibers are added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) (sample 6) and a medium composition (sample 12) not containing the above-mentioned substrate were prepared. Then, cultured human bone marrow-derived mesenchymal stem cells (C-12974, manufactured by Takara Bio Inc.) were inoculated at 80,000 cells / mL, and human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were inoculated at 100,000 cells / mL in the above-mentioned medium composition to which chitin nanofibers and chitosan nanofibers are added or in which no medium composition is added, and then the medium composition to which chitin nanofibers and chitosan nanofibers are added was dispensed into the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning Incorporated) in a manner of 1 mL per well. In addition, as a comparison object, it was dispensed into the wells of a 24-well flat-bottomed adhesion surface microplate (#3526, manufactured by Corning) in a manner of 1 mL per well. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 2 days. On the second day, the culture medium composition added with chitin nanofibers and chitosan nanofibers and the culture medium composition not added were moved to a 15mL tube from each well, and after centrifugation at 300xg for 3 minutes, the culture supernatant was removed. Next, 1mL of MEMα culture medium containing 17% FBS was added, and the culture medium composition added with chitin nanofibers and chitosan nanofibers was returned to the wells of a 24-well flat-bottomed ultra-low adhesion surface microplate (#3473, manufactured by Corning). For the unadded culture medium composition, after removing the mesenchymal stem cell proliferation medium 2 culture medium, 1 mL of MEMα culture medium containing 17% FBS was added to a 24-well flat-bottom adhesive surface microplate (#3526, manufactured by Corning). Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. On the first day, the culture medium composition added with chitin nanofibers and chitosan nanofibers and the unadded culture medium composition were transferred from each well to a 15 mL tube, centrifuged at 300xg for 3 minutes, and the culture supernatant was recovered. At this time, in order to evaluate the number of cells, 1 mL of ATP reagent (CellTiter-Glo (registered trademark) luminescent cell viability assay, manufactured by Promega) was added to each sample after the culture supernatant was recovered to suspend it, and after standing at room temperature for 10 minutes, Enspire (manufactured by Perkin Elmer) was used to measure the luminescence intensity (RLU value), and the luminescence value of the culture medium itself was subtracted to measure the number of living cells.Then, the bFGF contained in the recovered culture supernatant was quantified using a bFGF ELISA kit (#ELH-bFGF-1, manufactured by RayBiotech). 100 μL of standard or sample solution was added to the well and shaken at room temperature for 2.5 hours. Then, the solution was discarded, and 300 μL of 1x washing solution was added and removed. Repeat the above operation 4 times. Add 100 μL of 1x detection antibody and shake at room temperature for 1 hour. Discard the solution, add 300 μL of 1x washing solution and remove. Repeat the above operation 4 times. Add 100 μL of HRP-streptavidin solution and shake at room temperature for 45 minutes. Then, discard the solution, add 300 μL of 1x washing solution and remove. Repeat the above operation 4 times. Add 100 μL of TMB One-Step Substrate reagent and shake at room temperature in the dark for 30 minutes. Finally, 50 μL of stop solution was added and the absorbance at 450 nm was measured. The bFGF concentration contained in each sample was calculated by four-parameter logistic regression of the standard curve. In order to calculate the secretion amount per unit cell number, the calculated bFGF amount was divided by the luminescence intensity to calculate the relative value.

[0266] As a result, whether it is mesenchymal stem cells derived from human bone marrow or mesenchymal stem cells derived from human adipose, the relative value of bFGF secretion is increased in the case of 3D culture with a combination of a medium composition and a low adhesion plate added with chitin nanofibers and chitosan nanofibers, compared with the case of monolayer culture without the addition of a culture medium composition and an adhesion plate. The relative values ​​of mesenchymal stem cells derived from human bone marrow or mesenchymal stem cells derived from human adipose are shown in Tables 15 and 16.

[0267] [Table 15]

[0268]

[0269] [Table 16]

[0270]

[0271] (Test Example 10: TSG-6 Production Amount in 3D Culture of Human Bone Marrow-Derived and Human Umbilical Cord-Derived Mesenchymal Stem Cells Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0272] A medium composition (final concentration: 0.05% (w / v)) (sample 6) in which chitin nanofibers and chitosan nanofibers were added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) and a medium composition (sample 12) not containing the above-mentioned substrate were prepared. Then, cultured human bone marrow-derived mesenchymal stem cells (C-12974, manufactured by Takara Bio Inc.) were inoculated at 80,000 cells / mL and human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) were inoculated at 40,000 cells / mL in the above-mentioned medium composition to which chitin nanofibers and chitosan nanofibers were added or in which no medium composition was added, and then the medium composition to which chitin nanofibers and chitosan nanofibers were added was dispensed into the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning Incorporated) in a manner of 1 mL per well. In addition, as a comparison object, 1 mL was dispensed into the wells of a 6-well flat-bottom adhesive surface microplate (#3516, manufactured by Corning) or a 24-well flat-bottom adhesive surface microplate (#3526, manufactured by Corning) in a manner such that each well was 1 mL. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 2 days. On the second day, the culture medium composition to which chitin nanofibers and chitosan nanofibers were added and the culture medium composition not added were moved from each well to a 15 mL tube, centrifuged at 300 x g for 3 minutes, and the culture supernatant was removed. After removing the culture supernatant, 1 mL of mesenchymal stem cell proliferation medium 2 medium or mesenchymal stem cell proliferation medium 2 medium containing TNF-α at a final concentration of 10 ng / mL or 20 ng / mL (#210-TA, manufactured by R&D System) was added to a 15 mL tube, and the medium composition added with chitin nanofibers and chitosan nanofibers was returned to the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning). After removing the culture medium, 1 mL of mesenchymal stem cell proliferation medium 2 medium or mesenchymal stem cell proliferation medium 2 medium containing TNF-α at a final concentration of 10 ng / mL or 20 ng / mL was added to the 24-well flat-bottom adhesion surface microplate (#3526, manufactured by Corning) was added. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. On the first day, the culture medium composition to which chitin nanofibers and chitosan nanofibers were added and the culture medium composition without addition were transferred from each well to a 15 mL tube, and the culture supernatant was recovered after centrifugation at 300 xg for 3 minutes.At this time, in order to evaluate the number of cells, 1 mL of ATP reagent (CellTiter-Glo (registered trademark) luminescent cell viability assay, manufactured by Promega) was added to each sample after the culture supernatant was recovered and suspended. After standing at room temperature for 10 minutes, Enspire (manufactured by Perkin Elmer) was used to measure the luminescence intensity (RLU value), and the luminescence value of the culture medium itself was subtracted to measure the number of living cells. Then, ELISA was used to quantify the TSG-6 contained in the recovered culture supernatant. TSG-6 antibody (#sc-65886, manufactured by Santacruz) diluted to 10 μg / mL with 0.2M carbonate-bicarbonate buffer (pH 9.2) was added to a Maxisorp flat-bottom plate (#44-2404-21, manufactured by Thermo Fisher Scientific) at 50 μL / well and allowed to stand at 4°C for 24 hours. After 24 hours, add 300 μL of PBST solution in which Tween 20 (#P7949, manufactured by Sigma-Aldrich) was added to D-PBS(-) (#043-29791, manufactured by FUJIFILM WakoPure Chemical Corporation) at a final concentration of 0.05% (v / v), and then remove it. Repeat this operation 3 times. Add 100 μL of PBST solution containing 5% BSA (#A2153, manufactured by Sigma-Aldrich) and let it stand at room temperature for 30 minutes. After discarding the solution, add 300 μL of PBST solution and remove it. Repeat this operation 3 times. And, add 50 μL of TSG-6 (#2104-TS, manufactured by R&D Systems) and evaluation samples prepared for the standard curve to each well and let it stand at room temperature for 2 hours. After discarding the solution, add 300 μL of PBST solution and remove it. Repeat this operation 3 times. Add 50 μL of a solution of biotinylated anti-human TSG-6 antibody (#BAF2104, manufactured by R&D Systems) diluted to 5 μg / mL with PBST, and let stand at room temperature for 120 minutes. After discarding the solution, add 300 μL of PBST solution and remove it. Repeat this operation 3 times. Add 50 μL of a solution of streptavidin-HRP (#ab7403, manufactured by Abcam) diluted to 200 ng / mL with PBST solution, and let stand at room temperature for 30 minutes. After discarding the solution, add 300 μL of PBST solution and remove it. Repeat this operation 3 times. Add 100 μL of substrate solution (#52-00-03, manufactured by KPL), and let stand at room temperature for 15 minutes. Finally, add 100 μL of stop solution (#50-85-06, manufactured by KPL), and measure the absorbance at 450 nm.The TSG-6 concentration contained in each sample was calculated by four-parameter logistic regression of the standard curve. In order to calculate the secretion amount per unit cell number, the calculated TSG-6 amount was divided by the luminescence intensity to calculate a relative value.

[0273] As a result, compared with the case of monolayer culture using no added culture medium composition and adhesion plate, the relative value of TSG-6 secretion increased in the case of 3D culture of the culture medium composition and low adhesion plate combination of chitin nanofibers and chitosan nanofibers. In addition, for the increase rate when the control is compared with TNF-α treatment for each sample, compared with the case of monolayer culture using no added culture medium composition and adhesion plate, the relative value of TSG-6 secretion increased in the case of 3D culture of the culture medium composition and low adhesion plate combination of chitin nanofibers and chitosan nanofibers. The relative value of each sample of mesenchymal stem cells derived from human bone marrow and mesenchymal stem cells derived from human umbilical cord is shown in Table 17 and Table 18.

[0274] [Table 17]

[0275]

[0276] [Table 18]

[0277]

[0278] (Test Example 11: VEGF Production in Human Bone Marrow-Derived 3D Culture Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers)

[0279] A medium composition (final concentration: 0.05% (w / v)) (sample 6) in which chitin nanofibers and chitosan nanofibers are added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) and a medium composition (sample 12) not containing the above-mentioned substrate were prepared. Then, cultured human bone marrow-derived mesenchymal stem cells (C-12974, manufactured by Takara Bio Inc.) were inoculated in the above-mentioned medium composition added with chitin nanofibers and chitosan nanofibers or the medium composition not added in such a manner that 100,000 cells / mL were obtained, and then the medium composition added with chitin nanofibers and chitosan nanofibers was dispensed into a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning) in such a manner that 1 mL was added per well, and the medium composition not added was dispensed into the wells of a 24-well flat-bottom adhesion surface microplate (#3526, manufactured by Corning) in such a manner that 1 mL was added per well. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 2 days. On the second day, the culture medium composition added with chitin nanofibers and chitosan nanofibers and the culture medium composition not added were moved to a 15 mL tube from each well, centrifuged at 300xg for 3 minutes, and the culture supernatant was removed. Next, 1 mL of MEMα culture medium containing 17% FBS was added, and the culture medium composition added with chitin nanofibers and chitosan nanofibers was returned to the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning). For the unadded culture medium composition, after removing the mesenchymal stem cell proliferation culture medium 2 culture medium, 1 mL of MEMα culture medium containing 17% FBS was added to a 24-well flat-bottom adhesion surface microplate (#3526, manufactured by Corning). Each plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. On the first day, the culture medium composition added with chitin nanofibers and chitosan nanofibers and the culture medium composition not added were moved to 15mL tubes from each well, and after centrifugation at 300xg for 3 minutes, the culture supernatant was recovered. At this time, in order to evaluate the number of cells, 1mL of ATP reagent (CellTiter-Glo (registered trademark) luminescence cell viability detection, Promega company system) was added to each sample after the culture supernatant was recovered and suspended, and after standing at room temperature for 10 minutes, Enspire (Perkin Elmer company system) was used to measure the luminescence intensity (RLU value), and the luminescence value of the culture medium itself was subtracted, thereby measuring the number of living cells. Then, about the VEGF contained in the recovered culture supernatant, VEGF165 ELISA kit, Human (#ENZ-KIT156-0001, Enzo Life Sciences company system) was used for quantitative analysis.Add 100 μL of standard or sample solution to the well and shake at room temperature for 60 minutes. Then, discard the solution, add 300 μL of 1x washing solution, and remove. Repeat the above operation 3 times. Add 100 μL of VEGF detection antibody and shake at room temperature for 30 minutes. Discard the solution, add 300 μL of 1x washing solution, and remove. Repeat the above operation 3 times. Add 100 μL of VEGF conjugate (blue) and shake at room temperature for 30 minutes. Then, discard the solution, add 300 μL of 1x washing solution, and remove. Repeat the above operation 4 times. Add 100 μL of TMB solution and shake at room temperature for 30 minutes. Finally, add 100 μL of stop solution 2 and measure the absorbance at 450 nm. The VEGF concentration contained in each sample is calculated by four-parameter logistic regression of the standard curve. In order to calculate the secretion amount per unit number of cells, the calculated VEGF amount is divided by the luminescence intensity to calculate the relative value.

[0280] As a result, when the cells were cultured in the medium composition to which chitin nanofibers and chitosan nanofibers were added, the amount of VEGF produced per unit cell increased compared to when the cells were cultured in the medium composition without the addition of the medium composition.

[0281] [Table 19]

[0282]

[0283] (Test Example 12: Changes in stem cell marker expression of human umbilical cord-derived mesenchymal stem cells in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers or microcarriers)

[0284] A medium composition in which the sample 6 obtained above was added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) at a final concentration of 0.05% (w / v) was prepared. In addition, 360 mg of Corning low-concentration SynthemaxII microcarriers (manufactured by Corning, 3781) were weighed, hydrated with sterile distilled water, and immersed in 10 mL of ethanol for 30 minutes. Then, the ethanol was removed, washed twice with 30 mL of PBS (-), and finally washed with 10 mL of mesenchymal stem cell proliferation medium 2 medium. As a comparative control, the above-mentioned microcarriers were added to 10 mL of mesenchymal stem cell proliferation medium 2 medium to prepare a medium composition (sample 13). Next, 600,000 cells of cultured human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Inc.) were suspended in 40 mL of the above-mentioned mesenchymal stem cell proliferation medium containing nanofibers, filled into the antibody immobilized bag A (manufactured by NIPRO, 87-362) used as a culture bag, and then cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. In addition, 600,000 cells of cultured human umbilical cord-derived mesenchymal stem cells were suspended in 15 mL of mesenchymal stem cell proliferation medium containing microcarriers, and then transferred to a Corning 125 mL disposable rotating bottle and cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. After 1 day, the culture was continued for 2 days by using a Micro-Stir slow magnetic stirrer (WHEATON, W900701-B) to vibrate at 30rpm for 15 minutes and then let stand for 2 hours. After 3 days, the cells cultured in the antibody immobilized bag A and the disposable rotating bottle replaced half of the culture medium and continued to be cultured under the same conditions as above for 4 days. After 4 days, 1mL was taken out from 40mL and moved to a 1.5mL tube and centrifuged at 300xg for 3 minutes. After removing the supernatant, 350μL of RLT solution (RNeasy mini kit, QIAGEN, #74106) was added to make an RNA extraction solution. Then, after adding 350μL of 70% ethanol to the RNA extraction solution, it was added to the RNeasy purification column and centrifuged at 8000xg for 15 seconds. Then, 700μL of RW1 solution was added to the RNeasy purification column and centrifuged at 8000xg for 15 seconds. Next, 500 μL of RPE solution was added, and the mixture was centrifuged at 8000×g for 15 seconds. 500 μL of RPE solution was further added, and the mixture was centrifuged at 8000×g for 2 minutes. RNase-free solution was added to the RNA in the RNeasy purification column to elute the RNA.Then, cDNA was synthesized from the obtained RNA using PrimeScript RT reagent Kit (PerfectReal Time) (manufactured by Takara Bio Inc., #RR037A). Real-time PCR was performed using the synthesized cDNA and Premix EX Taq (Perfect Real Time) (manufactured by Takara Bio Inc., #RR039A) and Taq man probe (manufactured by Applied BioSystems). As Taq man probe (manufactured by Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs01053049_s1 was used for SOX2, Hs04399610_g1 was used for NANOG, Hs00607978s1 was used for CXCR4, and Hs99999905_m1 was used for GAPDH. Real Time PCR7500 was used as the instrument. The relative values ​​of the values ​​of each target gene corrected based on the values ​​of GAPDH were analyzed and calculated for comparison.

[0285] As a result, by using a culture medium composition comprising a mixture of chitin nanofibers and chitosan nanofibers, the mesenchymal stem cells derived from human umbilical cord were cultured in a culture bag, and the relative expression of the OCT4, SOX2, NANOG genes reflecting undifferentiated and the CXCR4 gene reflecting the mobility associated with homing was confirmed to rise. On the other hand, under the condition of using Corning low-concentration SynthemaxII microcarriers to culture, the obvious increase of the expression of these genes was not confirmed. The relative values ​​of each gene expression are shown in Table 20.

[0286] [Table 20]

[0287]

[0288] (Test Example 13: bFGF Production in 3D Culture of Human Adipose-Derived Mesenchymal Stem Cells Using a Mixture of Chitin Nanofibers and Chitosan Nanofibers or Microcarriers)

[0289] A medium composition in which the sample 6 obtained above was added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) at a final concentration of 0.05% (w / v) was prepared. In addition, 360 mg of Corning low-concentration SynthemaxII microcarriers (manufactured by Corning, 3781) were weighed, hydrated with sterile distilled water, and immersed in 10 mL of ethanol for 30 minutes. Then, the ethanol was removed, washed twice with 30 mL of PBS (-), and finally washed with 10 mL of mesenchymal stem cell proliferation medium 2 medium. As a comparative control, the above-mentioned microcarriers were added to 10 mL of mesenchymal stem cell proliferation medium 2 medium to prepare a medium composition (sample 13). Next, 600,000 cells of cultured mesenchymal stem cells derived from human fat (C-12977, manufactured by Takara Bio Inc.) were suspended in 40 mL of the above-mentioned mesenchymal stem cell proliferation medium containing nanofibers, filled into the antibody immobilized bag A (manufactured by NIPRO, 87-362) used as a culture bag, and then cultured in a CO2 incubator (37°C, 5% CO2) for 3 days in a static state. In addition, 600,000 cells of cultured mesenchymal stem cells derived from human fat were suspended in 15 mL of mesenchymal stem cell proliferation medium containing microcarriers, transferred to a Corning 125 mL disposable rotating bottle, and cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. After 1 day, the culture was continued for 2 days by shaking at 30 rpm for 15 minutes using a Micro-Stir slow magnetic stirrer (manufactured by WHEATON, W900701-B), and then standing for 2 hours. After 3 days, 1 mL was taken out from the 40 mL in the culture bag and the spinner bottle and transferred to a 1.5 mL tube, and centrifuged at 300 x g for 3 minutes. After centrifugation, the supernatant was removed and 2 mL of PBS (-) was added for washing. After washing, centrifugation was performed at 300 x g for 3 minutes, and after removing PBS (-), 1 mL of MEMα medium (FUJIFILM Wako Pure Chemical Corporation, 135-15175) containing 17% FBS was used for suspension, and then added to a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning Incorporated), and cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 1 day. After 1 day, the culture solution was taken out, transferred to a 1.5 mL tube, and centrifuged at 300 x g for 3 minutes, and the culture supernatant was recovered in a new 1.5 mL tube.At this time, in order to evaluate the number of cells, 1 mL of ATP reagent (CellTiter-Glo (registered trademark) luminescent cell viability assay, manufactured by Promega) was added to each sample after the culture supernatant was recovered to suspend it. After standing at room temperature for 10 minutes, Enspire (manufactured by Perkin Elmer) was used to measure the luminescence intensity (RLU value), and the luminescence value of the culture medium itself was subtracted to measure the number of living cells. Next, the bFGF contained in the recovered culture supernatant was quantified using bFGFELISA kit (#ELH-bFGF-1, manufactured by RayBiotech). 100 μL of standard or sample solution was added to the well and shaken at room temperature for 2.5 hours. Then, the solution was discarded, and 300 μL of 1x washing solution was added and removed. Repeat the above operation 4 times. Add 100 μL of 1x detection antibody and shake at room temperature for 1 hour. Discard the solution, and 300 μL of 1x washing solution was added and removed. Repeat the above operation 4 times. Add 100 μL of HRP-streptavidin solution and shake at room temperature for 45 minutes. Then, discard the solution, add 300 μL of 1x washing solution, and remove it. Repeat the above operation 4 times. Add 100 μL of TMB One-Step Substrate reagent and shake at room temperature in the dark for 30 minutes. Finally, add 50 μL of stop solution and measure the absorbance at 450 nm. The bFGF concentration contained in each sample is calculated by the four-parameter logistic regression of the standard curve. In order to calculate the secretion amount per unit number of cells, the calculated bFGF amount is divided by the luminescence intensity to calculate the relative value.

[0290] The results show that the relative value of the amount of bFGF secreted by human adipose-derived mesenchymal stem cells in 3D culture using a culture medium composition supplemented with chitin nanofibers and chitosan nanofibers increased compared to 3D culture using a culture medium composition supplemented with microcarriers.

[0291] [Table 21]

[0292]

[0293] (Test Example 14: Changes in stem cell marker expression of human adipose-derived mesenchymal stem cells in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers produced by simultaneous defibration or poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation)

[0294] Chitin powder and chitosan powder were mixed in a weight ratio of 1:4, and the mixture was nanofiberized at 200 MPa and 5 passes to prepare α-chitin / chitosan nanofibers (hereinafter, the mixture of chitin nanofibers and chitosan nanofibers produced by this method is sometimes referred to as "chitin / chitosan nanofibers prepared by simultaneous defibration". In addition, poly (1,4)-N-acetyl-β-D-glucosamine nanofibers with a specific degree of acetylation (about 50% in this test example) (hereinafter, sometimes referred to as "N-acetylglucosamine amount-adjusted nanofibers") were prepared by the following method: poly (1,4)-N-acetyl-β-D-glucosamine was heated and refluxed in a 30% sodium hydroxide aqueous solution for 4 hours to adjust the amount of N-acetylglucosamine to about 50%, and then it was nanofiberized at 200 MPa and 5 passes. The chitin / chitosan nanofibers and N-acetylglucosamine-amount-adjusted nanofibers thus obtained by simultaneous defibration were diluted with injection water (OTSUKADISTILLED WATER) to 1% (w / w), respectively, and dispersed by inversion mixing, and the aqueous suspension was sterilized in an autoclave at 121°C for 20 minutes (samples 14 and 15, respectively). A medium composition was prepared in which sample 14 or sample 15 obtained above was added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by TakaraBio Inc.) to a final concentration of 0.05% (w / v).

[0295] (1) Culture medium composition to which sample 14 was added (chitin / chitosan nanofibers prepared by simultaneous defibration: 0.05% (w / v))

[0296] (2) Culture medium composition to which sample 15 was added (N-acetylglucosamine amount adjusted (50%), nanofibers: 0.05% (w / v))

[0297] Next, the cultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were cultured in a culture dish in 2D to proliferate, and then inoculated in the above-mentioned medium composition with nanofibers added or without the medium composition added to 50,000 cells / mL, and then dispensed into the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning) in a manner of 1.2 mL per well. Each plate was cultured in a CO2 incubator (37°C, 5% CO2) under static conditions for 13 days. At the time of inoculation, cells were collected on the 8th and 13th days, and 300 μL of RLT solution (RNeasy mini kit (manufactured by QIAGEN, #74106) was added to prepare RNA extraction solution. After adding 300 μL of 70% ethanol to the RNA extraction solution, it was added to the RNeasy purification column and centrifuged at 8000×g for 15 seconds. Next, 600 μL of RW1 solution was added to the RNeasy purification column, and centrifuged at 8000×g for 15 seconds. Next, 500 μL of RPE solution was added, and centrifuged at 8000×g for 15 seconds. Further 500 μL of RPE solution was added, and centrifuged at 8000×g for 2 minutes. RNase-free solution was added to the RNA present in the RNeasy purification column to elute it. Then, cDNA was synthesized from the obtained RNA using PrimeScript RT reagent Kit (PerfectReal Time) (manufactured by Takara Bio Inc., #RR037A). The synthesized cDNA and Premix EX Taq (Perfect Real Time) (Takara Bio Inc., #RR039A), Taq man probe (Applied BioSystems) was used for real-time PCR. As Taq man probe (Applied BioSystems), Hs04260367_gH was used for OCT4, Hs04399610_g1 was used for NANOG, and Hs99999904_m1 was used for PPIA. Real Time PCR7500 was used as the instrument. For analysis, the relative value of each target gene corrected based on the value of PPIA was calculated and compared.

[0298] As a result, compared with the 2D culture conditions at the time of inoculation, by using a culture medium composition containing chitin / chitosan nanofibers or N-acetylglucosamine amount-adjusted nanofibers prepared by simultaneous defibration, mesenchymal stem cells derived from human adipose were cultured in 3D in a 24-well flat-bottom ultra-low adhesion surface microplate, and an increase in the relative expression levels of NANOG and OCT4 genes reflecting undifferentiation was confirmed. The relative values ​​of NANOG gene expression are shown in Table 22, and the relative values ​​of OCT4 gene expression are shown in Table 23.

[0299] [Table 22]

[0300]

[0301] [Table 23]

[0302]

[0303] (Test Example 15: Proliferation of human adipose-derived mesenchymal stem cells and changes in stem cell marker expression in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers or a mixture of chitin nanofibers and other polysaccharides)

[0304] Methylcellulose powder (1.0 g) was added to water (100 mL), stirred and suspended, and then sterilized in an autoclave (121° C., 20 minutes) to obtain a 1.0% (w / v) methylcellulose aqueous solution (8 mL). Sample 1 (2 mL) was added thereto and mixed by blowing and pipetting to prepare a mixture of 0.2% (w / v) chitin nanofibers and 0.8% (w / v) methylcellulose (sample 16).

[0305] Deacylated gellan gum powder (0.8 g) was added to water (100 mL) and stirred and dissolved, and then autoclaved (121° C., 20 minutes) to obtain a 0.8% (w / v) deacylated gellan gum aqueous solution. The deacylated gellan gum aqueous solution (12.5 mL) was mixed with D-PBS (+) (containing Ca, Mg) (27.5 mL) using a culture medium preparation kit (Nissan Chemical FCeM (registered trademark)-series preparation kit) to obtain a 0.25% (w / v) deacylated gellan gum divalent cation crosslinked structure dispersion (8 mL), to which sample 1 (2 mL) was added, and mixed by blowing and suction to prepare a mixture of 0.2% (w / v) chitin nanofibers and 0.2% (w / v) deacylated gellan gum (sample 17).

[0306] Sodium alginate powder (1.0 g) was added to water (50 mL) and stirred and dissolved, and then sterilized in an autoclave (121° C., 20 minutes) to obtain a 2.0% (w / v) sodium alginate aqueous solution. The sodium alginate aqueous solution (10 mL) was mixed with D-PBS (+) (containing Ca and Mg) using a culture medium preparation kit (Nissan Chemical FCeM (registered trademark)-series preparation kit) to obtain a 1.0% (w / v) alginic acid divalent cation cross-linked structure dispersion (8 mL), to which sample 1 (2 mL) was added, and mixed by blowing and suction to prepare a mixture of 0.2% (w / v) chitin nanofibers and 0.8% (w / v) alginic acid (sample 18).

[0307] Next, a medium composition was prepared in which samples 6, 16, 17, and 18 obtained above were added to a mesenchymal stem cell proliferation medium 2 medium (C-28009, manufactured by Takara Bio Inc.) at a final concentration of 0.05% (w / v). It should be noted that the final concentrations of chitin nanofibers and chitosan nanofibers or other polysaccharides (methylcellulose, deacylated gellan gum, alginic acid) in the prepared medium composition were as follows:

[0308] (1) Culture medium composition to which sample 6 was added (chitin nanofiber: 0.01% (w / v), chitosan nanofiber: 0.04% (w / v))

[0309] (2) Culture medium composition to which sample 16 was added (chitin nanofibers: 0.01% (w / v), methylcellulose: 0.04% (w / v))

[0310] (3) Culture medium composition to which sample 17 was added (chitin nanofibers: 0.01% (w / v), deacylated gellan gum: 0.04% (w / v))

[0311] (4) Culture medium composition to which sample 18 was added (chitin nanofibers: 0.01% (w / v), alginic acid: 0.04% (w / v))

[0312] Next, cultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were inoculated at a density of 13,333 cells / mL into a culture medium composition obtained by adding chitosan nanofibers or other polysaccharides (methylcellulose, deacylated gellan gum, alginate) to the above-mentioned chitin nanofibers, and then dispensed into the wells of a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, manufactured by Corning Incorporated) at a density of 1.2 mL per well. Each plate was cultured in a static state in a CO2 incubator (37°C, 5% CO2) for 14 days. At the time of inoculation (day 0) and on days 7 and 14 after inoculation, each culture medium was suspended and dispensed in 300 μL portions, and then 300 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay (manufactured by Promega), the cells were suspended and allowed to stand at room temperature for about 10 minutes, and then the luminescence intensity (RLU value) was measured using FlexStation3 (manufactured by Molecular Devices), and the luminescence value of the culture medium itself was subtracted to calculate the number of viable cells.

[0313] On the 7th and 14th days, the cells were collected and 300 μL of RLT solution (RNeasy mini kit (manufactured by QIAGEN, #74106) was added to prepare RNA extraction solution. After adding 300 μL of 70% ethanol to the RNA extraction solution, it was added to the RNeasy purification column and centrifuged at 8000×g for 15 seconds. Next, 600 μL of RW1 solution was added to the RNeasy purification column and centrifuged at 8000×g for 15 seconds. Next, 500 μL of RPE solution was added and centrifuged at 8000×g for 15 seconds. Further 500 μL of RPE solution was added and centrifuged at 8000×g for 2 minutes. RNase-free solution was added to the RNA present in the RNeasy purification column to elute it. Then, cDNA was synthesized from the obtained RNA using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by Takara Bio Inc., #RR037A). The synthesized cDNA and Premix EX Taq (Perfect Real Time) (Takara Bio Inc., #RR037A) were used. BioInc., #RR039A), Taq man probe (Applied Bio Systems) were used for real-time PCR. As Taq man probe (Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs04399610_g1 was used for NANOG, and Hs99999904_m1 was used for PPIA. Real Time PCR7500 was used as the instrument. For analysis, the relative value of each target gene corrected based on the value of PPIA was calculated and compared.

[0314] As a result, among the polysaccharides combined with α-chitin nanofibers, the culture medium composition containing a mixture with chitosan nanofibers has the highest proliferation ability of mesenchymal stem cells derived from human fat, and the expression ability of OCT4 gene and NANOG gene reflecting undifferentiation is high. Although the culture medium composition containing a mixture of chitin nanofibers and any one of the polysaccharides in methylcellulose, deacylated gellan gum, and alginic acid also proliferates mesenchymal stem cells derived from human fat, the expression of OCT4 gene and NANOG gene is confirmed to be hyperactive, but it is weaker than the mixture with chitosan nanofibers. The results of the number of living cells are shown in Table 24, the results of NANOG gene expression are shown in Table 25, and the results of OCT4 gene expression are shown in Table 26.

[0315] [Table 24]

[0316]

[0317] [Table 25]

[0318]

[0319] [Table 26]

[0320]

[0321] (Test Example 16: Proliferation, survival maintenance, and dispersibility of cell aggregates of HEK293-IFNβ cells in 3D culture using a mixture of chitin nanofibers and chitosan nanofibers, a mixture of chitin nanofibers and chitosan nanofibers produced by simultaneous defibration, or poly (1,4)-N-acetyl-β-D-glucosamine nanofibers having a specific degree of acetylation)

[0322] A culture medium composition was prepared in which the sample 1 obtained above was added to an EMEM culture medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum in a manner to a final concentration of 0.02% (w / v) or 0.1% (w / v). In addition, a culture medium composition was prepared in which the sample 6, 14 or 15 was added to an EMEM culture medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) fetal bovine serum in a manner to a final concentration of 0.02% (w / v). In addition, an unadded culture medium composition (sample 12) not containing the above-mentioned substrate was prepared. It should be noted that the final concentration of each nanofiber in the prepared EMEM culture medium containing nanofibers and 10% (v / v) fetal bovine serum is as follows:

[0323] (1) Culture medium composition to which sample 1 was added (chitin nanofiber: 0.02% (w / v), chitosan nanofiber: 0% (w / v))

[0324] (2) Culture medium composition to which sample 1 was added (chitin nanofiber: 0.1% (w / v), chitosan nanofiber: 0% (w / v))

[0325] (3) Culture medium composition to which sample 6 was added (chitin nanofiber: 0.02% (w / v), chitosan nanofiber: 0.08% (w / v))

[0326] (4) Culture medium composition to which sample 14 was added (chitin / chitosan nanofibers prepared by simultaneous fibrillation: 0.1% (w / v))

[0327] (5) Medium composition to which sample 15 was added (N-acetylglucosamine amount adjusted (50%), nanofibers: 0.1% (w / v))

[0328] Next, the cultured HEK293-IFMβ cells that stably produce human IFMβ were suspended in each of the above-mentioned culture medium compositions to a density of approximately 200,000 cells / mL, and then inoculated at 1.2 mL / well in a 24-well flat-bottom ultra-low adhesion surface microplate (Corning, #3473). The cells were cultured in a CO2 incubator (37°C, 5% CO2) in a static state for a maximum of 21 days, and the culture medium was replaced every 1 or 2 days. At the time of inoculation (day 0) and on days 7, 14, and 21 after inoculation, each culture medium was suspended and aliquoted into 300 μL portions, and then 300 μL of ATP reagent (CellTiter-Glo TM Luminescence cell viability assay, Promega), suspended, left to stand at room temperature for about 10 minutes, FlexStation3 (Molecular Devices) was used to measure the luminescence intensity (RLU value), subtracting the luminescence value of the culture medium itself to calculate the number of living cells. In addition, photographs were taken on the 8th, 15th, and 21st days after inoculation to confirm the dispersibility of the cell spherical mass.

[0329] As a result, the effects of proliferation and maintaining survival were confirmed under all conditions in which each substrate was added, compared with the condition without addition. In addition, when a culture medium composition containing chitin / chitosan nanofibers prepared by simultaneous defibration or nanofibers with an adjusted amount of N-acetylglucosamine was used, a higher cell density was shown compared with chitin nanofibers alone. In addition, through time observation, large cell aggregates were observed on the 8th day under the condition of no addition, and under the condition of chitin nanofibers alone (sample 1), the cell aggregates gradually increased and the number of aggregates decreased. It can be considered that further agglutination between cell aggregates occurred under these conditions. On the other hand, when a culture medium composition to which chitin nanofibers and chitosan nanofibers were added and a culture medium composition containing chitin / chitosan nanofibers prepared by simultaneous defibration were used, agglutination between cell aggregates was suppressed. In particular, when a culture medium composition containing nanofibers with an adjusted amount of N-acetylglucosamine was used, small cell aggregates still maintained a tendency to disperse at the 21st day. The results of the number of viable cells are shown in Figure 1 The results of the photo observation are shown in Figure 2 (Day 8), Figure 3 (Day 15), Figure 4 (Day 21).

[0330] Industrial Applicability

[0331] According to the present invention, high-quality adhesive cells (such as mesenchymal stem cells) can be efficiently produced. When the cells obtained by the present invention are stem cells, these cells are extremely high-quality stem cells that maintain characteristics such as undifferentiation and migration in an ideal state. Therefore, the stem cells (such as mesenchymal stem cells) obtained by the present invention can be preferably used to fill organs and tissues lost due to diseases, etc. In addition, the cell secretions obtained by the present invention can also be used for the treatment of various diseases. Therefore, it can be considered that the present invention is extremely useful in, for example, the field of regenerative medicine.

[0332] This application is based on Japanese Patent Application No. 2018-164042 (filing date: August 31, 2018) and Japanese Patent Application No. 2019-134058 (filing date: July 19, 2019) filed in Japan, and all the contents of the above applications are included in this specification.

Claims

1. A medium composition for suspension culture of adherent cells, comprising: (1) Chitin nanofibers; and (2) Chitosan nanofibers, in, The ratio of chitin nanofibers to chitosan nanofibers is chitin nanofibers: chitosan nanofibers = 1:3-6 by weight. N-acetylglucosamine accounts for more than 90% of the sugar units that make up chitin. The proportion of N-acetylglucosamine in the sugar units constituting chitosan is 30% or less.

2. The suspension culture medium composition according to claim 1, wherein Adherent cells are adherent cells that self-aggregate under suspension culture.

3. The suspension culture medium composition according to claim 1 or 2, wherein The adherent cells are stem cells.

4. The medium composition for suspension culture according to claim 3, wherein The stem cells are mesenchymal stem cells.

5. The suspension culture medium composition according to claim 3 or 4, wherein Suspension culture is used for the proliferation of stem cells and for maintaining the multipotency and migration of stem cells.

6. A method for culturing adherent cells, comprising the step of suspending and culturing the adherent cells in the following culture medium composition, wherein the culture medium composition comprises: (1) Chitin nanofibers; and (2) Chitosan nanofibers, in, The ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers=1:3-6 by weight. N-acetylglucosamine accounts for more than 90% of the sugar units that make up chitin. The proportion of N-acetylglucosamine in the sugar units constituting chitosan is less than 30%. The adhesive cells are mesenchymal stem cells, The concentration of the total nanofibers contained in the culture medium composition, namely the chitin nanofibers and the chitosan nanofibers, is 0.006 to 0.05% in terms of w / v.

7. The culture method according to claim 6, wherein: Suspension culture is used for the proliferation of mesenchymal stem cells and for the maintenance of their multipotency and migration.

8. The culture method according to claim 6 or 7, further comprising the following steps: In step (1), the suspension culture medium composition according to any one of claims 1 to 5 is further added without performing a treatment to peel the suspension cultured cells from the chitin nanofibers and chitosan nanofibers; and Step (2) The mixture obtained in step (1) is subjected to suspension culture.

9. A method for producing cell secretions, comprising the step of culturing adherent cells in suspension in the following medium composition, wherein the medium composition comprises: (1) Chitin nanofibers; and (2) Chitosan nanofibers, in, The ratio of chitin nanofibers to chitosan nanofibers in the culture medium composition is chitin nanofibers:chitosan nanofibers=1:3-6 by weight. N-acetylglucosamine accounts for more than 90% of the sugar units that make up chitin. The proportion of N-acetylglucosamine in the sugar units constituting chitosan is less than 30%. The adhesive cells are mesenchymal stem cells, The concentration of the total nanofibers contained in the culture medium composition, namely the chitin nanofibers and the chitosan nanofibers, is 0.006 to 0.05% in terms of w / v.

10. The method of claim 9, wherein: The concentration of serum in the medium composition is 0.05 to 2.0% by weight.

11. The method according to claim 9 or 10, wherein: The cell secretion is at least one selected from the group consisting of low molecular weight compounds, proteins, nucleic acids, and cell secretory vesicles.

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