TW202208614A - Liquid composition for functional enhancement of cells - Google Patents

Abstract

The present invention provides a liquid composition that is for functional enhancement of cells, and that contains: deacylated gellan gum or a salt thereof; and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and that can undergo crosslinking by means of divalent metal ions.

Description

Liquid composition for promoting cell function

The present invention relates to a liquid composition for promoting cell function by suspending and culturing cells in a liquid composition to improve cell function, and a method for promoting cell function using the same.

In the field of regenerative medicine, intensive research is being carried out on the use of pluripotent stem cells such as iPS cells (induced pluripotent stem cells, induced pluripotent stem cells) or ES cells (Embryonic stem cells, embryonic stem cells) to construct organ regeneration methods. However, the establishment of ES cells is accompanied by ethical issues, and iPS cells have also been recognized as having the risk of canceration or the problem of time-consuming culturing. Therefore, the method of using adult stem cells such as lobe stem cells and neural stem cells with relatively low risk of cancerization, precursor cells such as adipose precursor cells and myocardial precursor cells with a relatively short differentiation induction period, or chondrocytes is also explored. possibility.

Treatment using adult stem cells and the like requires a large amount of high-quality cells, and therefore, a culture method capable of efficiently producing high-quality cells has been sought. For example, it is known that mesenchymal stem cells can be proliferated relatively easily by monolayer culture in a petri dish (also called two-dimensional (2D) culture, etc.).

However, according to recent reports, there is a possibility that the undifferentiated or proliferative potential of mesenchymal stem cells may decrease with subculture in the 2D culture method in a petri dish, and there is also concern about chemotaxis including targeting. The function of mesenchymal stem cells such as sex or anti-inflammatory effect is reduced. Therefore, from the viewpoint of producing high-quality cells in the 2D culture method, which is a conventional method, measures such as limiting the number of passages during culture must be taken. Whether the quality of homogeneity is maintained is worrying. In this regard, it is reported that the expression of markers supporting pluripotency is improved by forming a cell mass of adult stem cells and culturing them in suspension (Patent Document 1). However, the formation of a cell mass is a necessary technique, and there is no description about the cultivation in the state of a single cell (single cell) that does not form the cell mass. It is technically difficult to form a cell mass of uniform size, and the effect of cell mass is still in the research stage. Therefore, a single cell is generally administered in current cellular medicines. In this case, the cell mass needs to be made into For a single cell, the complexity of the steps can be imagined. In addition, the morphology of leaf stem cells produced by the 2D culture method is a spindle cell line adhered to the plastic surface, which is quite different from the shape of cells originally existing in the living body. Furthermore, in the 2D culture method, trypsin or the like is used for enzymatic treatment, so the individualized frozen cells after trypsin treatment are considered to have a possibility that the functions of adhesion factors and the like on the cell surface may be reduced.

On the other hand, the primary cultured cells used for cell therapy are not only the above-mentioned stem cells but also terminally differentiated cells such as hepatocytes and the like. The hepatocytes cannot be proliferated by the 2D culture method, and if they adhere to the culture plate, the function and the survival rate will be drastically reduced. Therefore, the frozen hepatocytes are thawed and used immediately. At this time, it is suggested that not only the viability immediately after thawing is reduced, but also the cell function, especially the adhesion factor on the cell surface, is lost due to the enzyme treatment such as collagenase used to isolate hepatocytes from the liver. Indeed, most of the donor-derived hepatocytes prepared by collagenase treatment showed reduced adhesion to collagen-coated culture dishes.

Polysaccharides such as deacylated gellan gum (DAG) are assembled by metal cations (eg, divalent metal cations such as calcium ions), thereby forming a three-dimensional network structure (an amorphous structure) in water. If the cells are cultured in a liquid medium containing the three-dimensional network structure, the cells in the medium are captured by the three-dimensional network structure and will not settle, so the cells can be kept in a uniformly dispersed suspension state without shaking, rotating, etc. Culture (suspended stationary culture) was performed. In addition, it is reported that the above-mentioned three-dimensional network structure can be formed without substantially increasing the viscosity of the liquid medium, so the medium composition comprising the three-dimensional network structure is also excellent in operability in subculture and the like (Patent Document 2). ). [Prior Art Literature] [Patent Literature]

Patent Document 1: Japanese Patent Laid-Open No. 2018-23401 Patent Document 2: International Publication No. 2014/017513

[Problems to be Solved by Invention]

The purpose of the present invention is to provide a method for improving cell quality. [Technical means to solve problems]

In order to solve the above-mentioned problems, the inventors of the present invention have conducted intensive studies and found that, by using a liquid medium composition containing deacylated gellan gum and sodium alginate, the cells are cultured in a single-cell state without forming cell aggregates. Mesenchymal stem cells can increase the expression of undifferentiated or chemotactic-related genes and the secretion of specific cytokines of mesenchymal stem cells. In addition, the present inventors found that the adhesion of hepatocytes can be enhanced by culturing hepatocytes in the liquid medium composition. Based on these findings, the inventors of the present invention conducted further studies to complete the present invention.

That is, the present invention is as follows: [1] A liquid composition comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked via a divalent metal ion, or In its salt form, the liquid composition is used to promote cellular function. [2] The liquid composition according to [1], wherein the concentration of the deacylated gellan gum or its salt in the liquid composition is 0.002 to 0.1 (w/w/ v)%, the concentration of the acidic polysaccharide or its salt is 0.004-0.2(w/v)% in terms of free form, the mass ratio of the acidic polysaccharide or its salt to the deacylated gellan gum or its salt In terms of free body, it is 1 or more. [3] The liquid composition according to [1] or [2], wherein the acidic polysaccharide is any one selected from the group consisting of alginic acid, pectin, and pectic acid. [4] The liquid composition according to [3], wherein the acidic polysaccharide is alginic acid. [5] The liquid composition according to any one of [1] to [4], which further contains a metal cation. [6] The liquid composition according to [5], wherein the metal cation is calcium ion. [7] The liquid composition according to any one of [1] to [6], wherein the cells are mesenchymal stem cells, and the cell function to be promoted is selected from the group consisting of undifferentiated maintenance state, chemotaxis, and secreted factors At least one of the group consisting of secretory abilities. [8] The liquid composition according to [7], wherein the cellular function to be promoted is the ability to secrete a secretory factor. [9] The liquid composition according to [8], wherein the secreted factor is selected from the group consisting of TSG-6 (TNF-stimulated gene 6 protein, tumor necrosis factor-stimulated gene 6 protein), STC-1 (Stanniocalcin-1, stannyl) Calcitonin-1), ANG (Angiogenin, angiopoietin), EGF (Epidermal Growth Factor, epidermal growth factor), MCP-1 (Monocyte Chemotactic Protein-1, monocyte chemoattractant protein-1), ENA- 78 (epithelial-derived neutrophil-activating peptide 78, epithelial cell-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor, basic fibroblast growth factor), IL-6 (Interleukin-6, interleukin -6), IL-8 (Interleukin-8, interleukin-8), VEGF (Vascular endothelial growth factor, vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D, vascular endothelial growth factor-D) , TIMP (Tissue inhibitors of matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor, platelet-derived growth factor) and TGF-β (transforming growth factor-β, transforming growth factor-β) composed of at least one of the group. [10] The liquid composition according to any one of [1] to [6], wherein the cells are hepatocytes, and the cell function to be promoted is cell adhesion ability. [11] A method for promoting cell function, comprising culturing cells in a liquid composition comprising deacylated gellan gum or a salt thereof, and maintaining randomness in a divalent metal cation medium Acidic polysaccharides or their salts that are in a coiled state and can be cross-linked by divalent metal ions. [12] The method according to [11], wherein the concentration of deacylated gellan gum or its salt in the liquid composition is 0.002 to 0.1 (w/v) in terms of free form of deacylated gellan gum %, the concentration of the acidic polysaccharide or its salt is 0.004 to 0.2 (w/v)% in terms of free form, and the mass ratio of the acidic polysaccharide or its salt to the deacylated gellan gum or its salt is in free form. In body conversion, it is 1 or more. [13] The method according to [11] or [12], wherein the acidic polysaccharide is any one selected from the group consisting of alginic acid, pectin, and pectic acid. [14] The method according to [13], wherein the acidic polysaccharide is alginic acid. [15] The method according to any one of [11] to [14], which further contains a metal cation. [16] The method according to [15], wherein the metal cation is calcium ion. [17] The method according to any one of [11] to [16], wherein the cells are mesenchymal stem cells, and the cell function to be promoted is selected from the group consisting of undifferentiated maintenance state, chemotaxis, and ability to secrete secreted factors At least one of the group formed. [18] The method according to [17], wherein the cell function to be promoted is the ability to secrete a secreted factor. [19] The method according to [18], wherein the secreted factor is selected from TSG-6 (TNF-stimulated gene 6 protein, tumor necrosis factor-stimulated gene 6 protein), STC-1 (Stanniocalcin-1, Stanniocalcin-1) Calcium-1), ANG (Angiogenin, angiopoietin), EGF (Epidermal Growth Factor, epidermal growth factor), MCP-1 (Monocyte Chemotactic Protein-1, monocyte chemoattractant protein-1), ENA-78 ( Epithelial-derived neutrophil-activating peptide 78, bFGF (Basic fibroblast growth factor, basic fibroblast growth factor), IL-6 (Interleukin-6, interleukin-6) ), IL-8 (Interleukin-8, interleukin-8), VEGF (Vascular endothelial growth factor, vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D, vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor) and TGF-β (transforming growth factor-β, transforming growth factor-β) at least one of them. [20] The method according to any one of [11] to [16], wherein the cells are hepatocytes, and the cell function to be promoted is cell adhesion ability. [Effect of invention]

According to the present invention, specific functions of cells can be promoted. For example, it can improve the adhesion (or survival rate) of hepatocyte transplantation, or promote the function of mesenchymal stem cells (such as undifferentiated state, chemotaxis, secretory factor secretion ability caused by repeated passage in 2D culture, etc.) ) to improve its quality.

Hereinafter, the present invention will be described in detail.

1. Liquid composition The present invention provides a liquid composition (hereinafter sometimes referred to as "liquid composition of the present invention"), which comprises deacylated gellan gum or a salt thereof, and is maintained in a divalent metal cation medium. Acidic polysaccharides or their salts that are in a random coil state and can be cross-linked by divalent metal ions, the liquid composition is used to promote cell function.

The cell in the present invention refers to the most basic unit constituting an animal or a plant, and has a cytoplasm and various organelles as its elements inside the cell membrane. In this case, the DNA-encapsulating nucleus may or may not be included inside the cell. For example, cells derived from animals in the present invention include germ cells such as sperm or eggs, somatic cells constituting living organisms, stem cells, precursor cells, cancer cells isolated from living organisms, and cells isolated from living organisms that obtain immortalization ability. Stable cells (cell lines) maintained in vitro, cells isolated from organisms and subjected to artificial genetic modification, cells isolated from organisms and subjected to artificial nuclear exchange, etc. Examples of somatic cells constituting a living body are not limited to the following, and include fibroblasts, osteocyte cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes ), osteocytes, osteocyte cells, coat cells, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells , neural lineage cells, glial cells, neurons, oligodendritic cells, microglial cells, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g. smooth or skeletal muscle cells), pancreatic beta cells, Melanocytes, hematopoietic precursor cells, and mononuclear cells, etc. The somatic cells include, for example, from skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, blood vessels Cells collected from any tissue, blood, heart, eye, brain or nerve tissue. The so-called stem cells refer to cells that have both the ability of self-replication and the ability to differentiate into cells of various other systems. Examples are not limited to the following, including: embryonic stem cells (ES cells), embryonic tumor cells, embryonic reproductive stem cells, artificial Stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, etc. The so-called precursor cells refer to cells in the middle stage of differentiation from the above-mentioned stem cells to specific somatic cells or germ cells. The so-called cancer cells refer to cells derived from autologous cells that have acquired unlimited proliferation potential. The so-called cell line refers to the cell that obtains infinite proliferation potential by artificial manipulation in vitro. As an example, it is not limited to the following, including: CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cell line) ), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7/TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 /1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero, etc.

The cells to be cultured in the liquid composition of the present invention can be arbitrarily selected from the cells described above. Cells can be harvested directly from animals or plants. Cells can also be induced from animals or plants, grown, or transformed and harvested by subjecting them to specific treatments. In this case, the treatment may be performed in vivo or in vitro. Examples of animals include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals, and the like. Examples of mammals are not limited, but include rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypus, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, Elephant, tamarin, squirrel monkey, rhesus macaque, chimpanzee and humans. The plant is not particularly limited as long as the collected cells or tissues can be cultured in liquid. For example, plants that produce crude drugs (such as saponins, alkaloids, berberine, scopolamine, phytosterols, etc.) (such as medicinal ginseng, periwinkle, scopolamine, Coptis chinensis, belladonna, etc.), or Plants (such as blueberries, safflower, madder, saffron, etc.) used as pigments or polysaccharides (such as anthocyanins, safflower pigments, madder pigments, saffron pigments, flavonoids, etc.) as cosmetics, food raw materials, or production of pharmaceuticals The original plant, etc., but not limited to these. In a preferred form, the cells may be mesenchymal stem cells or hepatocytes.

In the present specification, the so-called mesenchymal stem cells refer to adult stem cells with self-replication ability and multi-differentiation potential to differentiate into various mesenchymal cells. The source tissue of the leaf stem cells to which the liquid composition of the present invention is applied is not particularly limited, and may be any one such as bone marrow, fat, and umbilical cord.

The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked by a divalent metal ion.

The liquid composition of the present invention contains deacylated gellan gum or its salt, and an acidic polysaccharide or its salt which maintains a random coil state in a divalent metal cation medium and can be cross-linked by the divalent metal ion , the mesenchymal stem cells can be cultured in a suspended state (preferably in a suspended state).

The term "suspended" in the present invention refers to a state in which cells do not adhere to the culture vessel (non-adherent). Furthermore, in the present invention, when cells are cultured in the liquid composition, the cells are uniformly dispersed in the liquid composition without external pressure or vibration on the liquid composition, or shaking or rotating operations of the liquid composition. Dispersed and in a suspended state, this state is called "suspended stationary", and culturing cells in this state is called "suspended stationary culture". In addition, the period of time that can be suspended under "suspending and standing" includes more than 5 minutes (for example, at least 5 to 60 minutes), more than 1 hour (for example, 1 hour to 24 hours), 24 hours or more (for example, 1 day to 21 days), More than 48 hours, more than 7 days, etc., as long as the suspension is maintained, there is no limit to the length of time.

In a preferred form, the liquid composition of the present invention can suspend the cells at least at a certain temperature within the temperature range (eg, 0-37° C.) that enables the cells to be cultured in a non-frozen state. The liquid composition of the present invention can be at least a certain temperature in a temperature range of preferably 1 to 30°C, more preferably at least a temperature in a temperature range of 15 to 30°C, and still more preferably a temperature of 22 to 28°C At least a certain temperature in the range, more preferably at least a certain temperature in the temperature range of 24 to 26°C, preferably at least 25°C, the cell suspension is allowed to stand.

Whether it can be suspended or not can be evaluated by the following methods. For example, the cells of the culture object are uniformly dispersed in the liquid composition of the evaluation object at a concentration of 2×10 ⁴ cells/ml, and 10 ml is injected into a 15 ml conical tube. , stand at the desired temperature (eg, 25°C, 37°C) for at least 5 minutes (eg, 1 hour, 24 hours, 48 hours, 7 days), and observe whether the cells remain in suspension. When more than 70% of all cells are in a suspended state, it can be concluded that the suspended state is maintained. The evaluation can also be performed with polystyrene beads (500-600 μm in size, manufactured by Polysciences Inc.) instead of cells.

The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked by a divalent metal ion. The liquid composition of the present invention contains deacylated gellan gum or its salt, and an acidic polysaccharide or its salt which maintains a random coil state in a divalent metal cation medium and can be cross-linked by the divalent metal ion , cells can be cultured while maintaining good viability. In a preferred form, the liquid composition of the present invention is formed by including deacylated gellan gum or its salt, maintaining a random coil state in a divalent metal cation medium, and being cross-linked by divalent metal ions. Acidic polysaccharides or their salts have the property that cells can be cultured in a suspended state (preferably, suspended in a static state) (the effect of maintaining the suspended state of cells or tissues).

Deacylated gellan gum is a tetramolecular sugar of 1-3-bonded glucose, 1-4-bonded glucuronic acid, 1-4-bonded glucose and 1-4-bonded rhamnose as structural units The linear polymer polysaccharide is a polysaccharide represented by the following general formula (I) (here, R ₁ and R ₂ are both hydrogen atoms, and n is an integer of 2 or more). Wherein, R ₁ may contain a glycerol group, and R ₂ may contain an acetyl group, and the content of the acetyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

[hua 1]

Deacylated gellan gum can be produced by the following methods: culturing the microorganisms producing gellan gum in a fermentation medium, performing alkali treatment on the mucous membranes produced in vitro, and adding 1-3 bonded glucose residues on the The bound glycerol group and acetyl group are recovered after deacetylation, and are made into powder after drying, pulverization and other steps. As the purification method, for example, liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography methods, gel filtration chromatography using Sephadex LH-20, etc., adsorption chromatography using activated carbon, silica gel, etc., or Adsorption and desorption of active substances by thin-layer chromatography, high-performance liquid chromatography using reversed-phase column, etc. are used alone, or a combination of these methods is used in any order, or a certain method is used repeatedly. method whereby impurities can be removed for purification. The examples of microorganisms that produce gellan gum are not limited to these, and examples thereof include Sphingomonas elodea and microorganisms obtained by genetic modification of the microorganisms.

Phosphorylated ones can also be used for deacylated gellan gum. This phosphorylation can be performed by a known method.

The hydroxyl group corresponding to R ₁ and/or R ₂ of the compound represented by the general formula (I) is substituted with a monosaccharide residue such as C _1-3 alkoxy, C _1-3 alkylsulfonyl, glucose or fructose , oligosaccharide residues such as sucrose, lactose, amino acid residues such as glycine, arginine, and the like can also be used in the present invention. In addition, a crosslinking agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) can also be used to crosslink the deacylated gellan gum.

Examples of 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; tetraethylammonium, tetrabutylammonium, etc. Quaternary ammonium salts such as ammonium, methyl tributyl ammonium, cetyl trimethyl ammonium, benzyl methyl hexyl decyl ammonium, choline; and pyridine, triethylamine, diisopropylamine, ethanolamine , salts of organic amines such as diethanolamine, tromethamine, meglumine, procaine, chloroprocaine; and salts of amino acids such as glycine, alanine, valine, etc.

The weight average molecular weight of the deacylated gellan gum or its salt is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and still more preferably 1,000,000 to 10,000,000. The molecular weight can be measured, for example, by pullulan conversion based on gel permeation chromatography (GPC).

As the deacylated gellan gum or its salt, commercially available products such as "KELCOGEL (registered trademark of CP Kelco Corporation) CG-LA" manufactured by Sanjing Co., Ltd., manufactured by San-Ei Gen F.F.I. "KELCOGEL (registered trademark of CP Kelco)", etc.

As a medium of divalent metal cations (such as calcium ions, magnesium ions, barium ions, copper ions, iron ions, zinc ions, tin ions, lead ions, etc., preferably calcium ions) to maintain the random coil state, and can Acidic polysaccharides or their salts cross-linked by divalent metal ions include alginic acid (including oligomeric alginic acid (sometimes also referred to as "alginic acid oligosaccharide"), pectin, pectic acid, the and the like, preferably alginic acid (including oligomeric alginic acid) or a salt thereof.

Alginic acid is a polysaccharide having a structure in which α1-4-bonded L-glucuronic acid and β1-4-bonded D-mannuronic acid are linearly bonded to two uronic acids.

Alginic acid or a salt thereof can be extracted and purified from brown algae such as kelp and wakame by performing an ion exchange reaction with respect to the carboxyl group of alginic acid. The alginic acid in the algae forms an insoluble salt with multivalent cations such as calcium ions, so it is ion-exchanged with Na to become water-soluble sodium alginate, which is extracted from the algae. Furthermore, by adding acid to the aqueous solution of sodium alginate, insoluble alginic acid is coagulated and precipitated, and the coagulated and precipitated alginic acid is isolated, whereby purified alginic acid can be obtained.

Examples of 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; tetraethylammonium, tetrabutylammonium, etc. Quaternary ammonium salts such as ammonium, methyl tributyl ammonium, cetyl trimethyl ammonium, benzyl methyl hexyl decyl ammonium, choline; and pyridine, triethylamine, diisopropylamine, ethanolamine , salts of organic amines such as diethanolamine, tromethamine, meglumine, procaine, chloroprocaine; and salts of amino acids such as glycine, alanine, valine, etc. In the present invention, sodium alginate is preferably used from the viewpoint of solubility in water.

The weight average molecular weight of alginic acid or its salt is, for example, 300 to 5,000,000, preferably 300 to 1,000,000, more preferably 300 to 500,000, further preferably 300 to 100,000, and most preferably 300 to 10,000. The molecular weight can be measured, for example, by pullulan conversion based on gel permeation chromatography (GPC).

As alginic acid (including oligomeric alginic acid) or its salt, commercially available products such as the following products can also be used. KIMICA Co., Ltd.: Kimica Algin SeriesIL-2, IL-6, I-1, I-3, I-5, I-8, ULV-L3, ULV-L5, ULV-1, ULV-3, ULV-5, ULV-20 , ULV-L3G, IL-6G, I-1G, I-3G, IL-6M, BL-2, BL-6, B-1, B-3, B-5, B-8, SKAT-ONE, SKAT -ULV Algitex series LL, L, M, H Kikkoman Biochemifa Co., Ltd.: Duck Algin NSPH2R, NSPHR, NSPMR, NSPLR, NSPLLR Sanjing Co., Ltd.: Sukohgin, San Algin Hokkaido Mitsui Chemicals Co., Ltd.: Alginate Oligosaccharide ALGIN

Deacylated gellan gum, and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions can be formed by intracyclic or exocyclic isomerization. The isomers, geometric isomers, tautomers or mixtures of geometric isomers, or mixtures of these exist. Deacylated gellan gum, and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions, whether or not formed by isomerization, have asymmetric centers. Where appropriate, it may exist as resolved optical isomers or as a mixture comprising these in any ratio.

Deacylated gellan gum or its salts, acid polysaccharides or their salts which maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions or their salts via metal cations ( For example, divalent metal cations such as calcium ions) aggregate to form a three-dimensional network structure (amorphous structure). It is known that polysaccharides form microgels via metal cations (for example, Japanese Patent Laid-Open No. 2004-129596 ), and this microgel is also included in one of the forms of the above-mentioned amorphous structures. Regarding deacylated gellan gum or its salts, and acidic polysaccharides or salts thereof that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions, or their salts assembled by metal cations, As one form, a film-shaped structure can be mentioned. The liquid composition of the present invention comprises an acidic polysaccharide which is maintained in a random coil state in a divalent metal cation medium and can be cross-linked through the divalent metal ion by the deacylation gellan gum or its salt or its salt. A salt is a three-dimensional network structure (amorphous structure) formed by aggregation of metal cations (eg, divalent metal cations such as calcium ions). If the cells are suspended in the liquid composition of the present invention and cultured, the cells suspended in the liquid composition will be captured by the three-dimensional network structure without sedimentation. Therefore, the cells can be suspended in a suspended state without shaking, rotating, or the like. The culture is carried out in a state of uniform dispersion (suspended stationary culture). The liquid composition of the present invention preferably contains the above-mentioned three-dimensional network structure (unshaped structure) in a uniformly dispersed state.

In a preferred form, the formation of the above-mentioned three-dimensional network structure (unshaped structure) is not particularly limited as long as the liquid composition of the present invention is a plastic fluid and has fluidity. In particular, the viscosity of the liquid composition does not exceed 10,000 mPa·s. In this case, the viscosity of the liquid composition at 25°C is 5000 mPa·s or less, preferably 1000 mPa·s or less, more preferably 100 mPa·s or less.

The viscosity of a liquid composition can be measured by the method described in the Example mentioned later, for example. Specifically, E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, conical rotor: standard rotor 1°34'×R24, 100 rpm) for measurement.

The liquid composition of the present invention may also include "dearylated gellan gum or a salt thereof, and an acidic polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked by a divalent metal ion or its salt. polysaccharides other than salts or their salts. The polysaccharides are preferably acidic polysaccharides having anionic functional groups. The acidic polysaccharide is not particularly limited as long as it has an anionic functional group in its structure, and for example, it has uronic acid (eg, glucuronic acid, iduronic acid, galacturonic acid, and mannuronic acid). The polysaccharides, the polysaccharides with a part of the structure having a sulfate group or a phosphate group, or the polysaccharides with the structure of both, are not only naturally obtained polysaccharides, but also include polysaccharides produced by microorganisms, genetically engineered polysaccharides. Produced polysaccharides, or artificially synthesized polysaccharides using enzymes. More specifically, hyaluronic acid, natural gellan gum, rhamnose gum, Dingyou gum, Sanxian gum, carrageenan, Sanxian gum, hexuronic acid, fucoidan, pectin, Pectic acid, pectin ester acid, heparin sulfate, heparin, heparan sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamnose sulfate, or salts of these.

The concentration of deacylated gellan gum or its salt in the liquid composition of the present invention (conversion of free form of deacylated gellan gum) is, for example, 0.002 to 0.1 (w/v)%, preferably 0.002 to 0.009 ( w/v)%, more preferably 0.003 to 0.009(w/v)%, still more preferably 0.0033 to 0.0066(w/v)%.

Concentration (free body conversion) of acidic polysaccharides (eg alginic acid) or salts thereof that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions in the liquid composition of the present invention For example, it is 0.004-0.2(w/v)%, preferably 0.004-0.02(w/v)%, more preferably 0.004-0.015(w/v)%, and still more preferably 0.005-0.015(w/v)% %, more preferably 0.0066 to 0.0133 (w/v)%.

From the viewpoint of sufficiently securing the effect of suspending cells, the concentration of deacylated gellan gum or its salt is preferably 0.002 (w/v)% or more, more preferably 0.003 (w/v)% or more. On the other hand, if the concentration is too high, the suspending action may become stronger, resulting in a decrease in the cell recovery rate or a decrease in the workability of the medium itself. Therefore, it is preferably 0.1 (w/v)% or less. Preferably, it is 0.009(w/v)% or less. Maintenance of random wires in a divalent metal cation medium from the viewpoint of ensuring that the effect of maintaining cells in suspension quickly disappears by shear force (the effect of maintaining cells in suspension against vulnerability to shear force) The concentration of acidic polysaccharides (such as alginic acid) or their salts that can be cross-linked by divalent metal ions in an aggregated state is preferably 0.004 (w/v)% or more, preferably 0.005 (w/v)% above. On the other hand, if the concentration is too high, there is a possibility of gelation, so it is preferably 0.2 (w/v)% or less, more preferably 0.02 (w/v)% or less, more preferably 0.015 ( w/v)% or less.

Regarding the deacylated gellan gum or its salts contained in the liquid composition of the present invention, and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions ( For example, the mass ratio of alginic acid) or its salt (converted to free form), from the viewpoint of achieving the property of rapidly disappearing the effect of maintaining the suspended state of cells by shear force, is relative to deacylated gellan gum or its salt. 1 part by mass, the acid polysaccharide (such as alginic acid) or its salt that will maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions is set to 1 part by mass or more, preferably 2 parts by mass or more. In one form, relative to 1 part by mass of deacylated gellan gum or its salt, an acidic polysaccharide (such as an acid polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions) alginic acid) or its salt is, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, and more preferably 1 to 2 parts by mass.

In addition, the compound concentration in a liquid composition can be calculated according to the following formula.

Concentration [(w/v)%] = mass of compound (g) / volume of liquid composition (ml) × 100

The liquid composition of the present invention contains deacylated gellan gum or its salt in the above-mentioned content, and an acidic polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked through the divalent metal ion (such as alginic acid) or its salts, and exert the effect of maintaining the good viability of the cultured cells. The liquid composition of the present invention contains deacylated gellan gum or its salt in the above-mentioned content, and an acidic polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked through the divalent metal ion (such as alginic acid) or its salt, and play the effect of maintaining the suspended state of cells.

The liquid composition of the present invention contains deacylated gellan gum or its salt in the above-mentioned content, and an acidic polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked through the divalent metal ion (such as alginic acid) or its salts, and it also has the property of rapidly losing the effect of maintaining the suspended state of cells by shearing force such as pipetting or filter filtration (the effect of maintaining the suspended state of cells is vulnerable to shearing force). sex).

The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide (such as alginic acid) that maintains a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions A three-dimensional network structure (amorphous structure) formed by the aggregation of metal cations (such as divalent metal cations such as calcium ions) or its salts, thereby producing the effect of maintaining the suspended state of cells, but due to the inclusion of divalent metal cations in the Acidic polysaccharides (such as alginic acid) or their salts that maintain the state of random coils in the medium and can be cross-linked by divalent metal ions, and the three-dimensional network structure becomes fragile in response to chelating agents or shearing forces. The structure is easily damaged by shear force such as pipetting or filter filtration, and the effect of maintaining the suspension of cells is quickly lost. Deacylated gellan gum has structural units with a relatively linear structure. By bunching a plurality of deacylated gellan gum chains in a liquid composition, a compact and stable three-dimensional network structure is formed. Therefore, the three-dimensional The network structure is not easily destroyed by chelating agents, pipetting or filter filtration, etc. On the other hand, if the liquid composition is added to the liquid composition by L-glucuronic acid containing α1-4 bonds and D bonded to β1-4 -Mannuronic acid two uronic acids with a relatively bulky structure that maintains a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions. Acidic polysaccharides (such as alginic acid) or its The salt suppresses the bunching of the deacylated gellan gum and makes the three-dimensional network structure vulnerable to shearing forces such as pipetting or filter filtration, but this theory is not particularly limited.

As described above, in the liquid composition of the present invention, the deacylated gellan gum or its salt, and the acid polyamide that maintains a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions Saccharides (such as alginic acid) or their salts are assembled by metal cations (such as divalent metal cations such as calcium ions) in the liquid composition to form a three-dimensional network structure (unshaped structure). Therefore, the liquid composition of the present invention contains Metal cations, such as divalent metal cations (calcium ion, magnesium ion, zinc ion, iron ion and copper ion, etc.), preferably calcium ion. The metal cation can be used in combination of, for example, calcium ion and magnesium ion, calcium ion and zinc ion, calcium ion and iron ion, and calcium ion and copper ion. The combination can be appropriately determined by the manufacturer. The concentration of metal cations in the liquid composition of the present invention is 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM, but not limited thereto.

The destruction of the three-dimensional network structure (loss of the property of maintaining the suspended state of cells or tissues) by shear force such as pipetting or filter filtration is a reversible reaction. The reason for this is that the fragments of the three-dimensional network structure (amorphous structure) destroyed by shear force are reassembled through metal cations (such as divalent metal cations such as calcium ions), and the three-dimensional network structure (amorphous structure) is formed again. structure).

The liquid composition of the present invention comprises a medium for cell culture. The liquid composition of the present invention can maintain a random coil state by mixing a medium for cell culture, deacylated gellan gum or a salt thereof, and a divalent metal cation medium, and can be exchanged through divalent metal ions. It is prepared by mixing together the acidic polysaccharides (such as alginic acid) or its salts.

Components that can be added to the medium when culturing cells may be further added to the liquid composition of the present invention. For example, fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, acetone Sodium, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices or various cell adhesion molecules, etc. . Examples of cytokines added to the liquid composition of the present invention include interleukin-1 (IL-1), interleukin-2 (IL-2), and 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), monocytogenes 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), tumor suppressin M(OM), erythropoietin (EPO), thrombopoietin (TPO), etc., but not limited to these.

Examples of hormones to be added to the liquid composition of the present invention include melanin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Müllerian hormone, lipid Catenin, corticotropin, pro-angiotensin and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcistatin, cholecystokinin, corticotropin, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin, ghrelin, human chorionic gonadotropin, human placental lactogen, growth hormone, statin, insulin, insulin-like growth factor, leptin, luteinizing hormone hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydrogenase Androstone, Androstenedione, Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone, Calcitriol, Calcidiol, Prostaglandin, Leukotriene, Prostacyclin, Thrombin, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin and enkephalin, but not limited to these.

As the growth factor added to the liquid composition of the present invention, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP- 1α), epithelial cell growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8 or 9 (FGF-1, 2, 3, 4, 5, 6, 7 , 8, 9), nerve cell growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease linker I, proteinase link II, platelet-derived growth factor (PDGF), cholinergic Differentiation factor (CDF), chemokine, Notch ligand (Delta1, etc.), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF) , insulin-like growth factor binding protein (IGFBP), pleiotrophin (Pleiotrophin), etc., but not limited to these.

In addition, those obtained by artificially remodeling the amino acid sequences of these cytokines or growth factors by gene recombination technology can also be added. Examples thereof include IL-6/soluble IL-6 receptor complex, Hyper IL-6 (fusion protein of IL-6 and soluble IL-6 receptor), and the like.

Examples of various extracellular matrices or various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nidogen, tenascin, Thrombospondin, von Willebrand factor, osteomodulin, fibrinogen, various elastin, various proteoglycans, various cadherins, desmoglein, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, polyglucosamine , agarose gel, hyaluronic acid, alginic acid gel, various hydrogels, and fragmented fragments of these.

Examples of antibiotics added to the liquid composition of the present invention include sulfonamide, penicillin, phenacillin, dimethicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, Nafcillin, Ampicillin, Penicillin, Amoxicillin, Cyclohexillin, Carbenicillin, Ticarcillin, Piperacillin, Azlocillin, Mezlocillin, Mecillin, Amdinocillin, Cephalosporins and its derivatives, oxolinic acid, amfloxacin, temafloxacin, nalidixic acid, pyrrole acid, ciprofloxacin, cinoxacin, norfloxacin, pefloxacin, rosoxa Star, Ofloxacin, Enoxacin, Pipemidic Acid, Sulbactam, Clavulanic Acid, β-Bromopenicillic Acid, β-Chloropenicillanic Acid, 6-Acetylmethylene-Penicillanic Acid , cefoxazole, sutacillin, amdrocillin and sulbactam formaldehyde hydrate ester, tazobactam, aztreonam, sulfamethoxazole, isosulfamide, nocardiazone, Meta-carboxyphenol, methyl phenylacetamidophosphonate, chlorotetracycline, oxytetracycline, tetracycline, chlortetracycline, doxycycline, metacycline, and minocycline.

In a preferred form, the liquid composition of the present invention contains metal cations (such as divalent metal cations (calcium ion, magnesium ion, zinc ion, iron ion and copper ion, etc.), preferably calcium ion). Deacylated gellan gum or its salts, and acidic polysaccharides (such as alginic acid) or its salts that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions When the liquid composition of cations is mixed, the metal cations in the liquid composition (eg, divalent metal cations such as calcium ions) aggregate to form a three-dimensional network structure (unshaped structure). With regard to the concentration of metal cations (preferably calcium ions) in the liquid composition, as long as the concentration is sufficient to maintain the random coil state in the deacylated gellan gum or its salt, and in the divalent metal cation medium, and can be obtained via Divalent metal ions cross-linked acidic polysaccharides (such as alginic acid) or its salts form a three-dimensional network structure (amorphous structure) through the aggregation of the metal cations, which is not particularly limited, for example, 0.1 mM to 300 mM, more Preferably 0.5 mM to 100 mM. The liquid composition containing the metal cation, the deacylated gellan gum or its salt, and the acid polysaccharide that maintains a random coil state in a divalent metal cation medium and can be cross-linked by the divalent metal ion (for example, alginic acid) or its salts are mixed, and the liquid composition without the metal cations can also be mixed with deacylated gellan gum or its salts, and in a divalent metal cation medium to maintain a random coil state, The acidic polysaccharides (eg, alginic acid) or their salts cross-linked by divalent metal ions can be mixed, and then an aqueous solution containing the metal cations prepared separately is added to the mixed solution.

In addition to the above-mentioned composition, the liquid composition of the present invention may also contain various components having the effect of prolonging cell life during cell culture. Examples of the component include saccharides (excluding polysaccharides) (for example, monosaccharides (such as glucose) and disaccharides), antioxidants (for example, SOD, vitamin E, glutathione, and polyphenols), hydrophilic polymers compounds (eg, polyvinylpyrrolidone), chelating agents (eg, EDTA), sugar alcohols (eg, mannitol, sorbitol), glycerol, etc., but not limited to these. In one form, the liquid composition of the present invention contains saccharides (excluding polysaccharides) (such as monosaccharides (glucose, etc.), disaccharides), antioxidants (such as SOD, vitamin E, glutathione, polysaccharides, etc.) phenol), hydrophilic polymers (eg polyvinylpyrrolidone), chelating agents (eg EDTA), sugar alcohols (eg mannitol, sorbitol) and at least one compound from the group consisting of glycerol.

In one form, the liquid composition of the present invention does not contain saccharides (excluding polysaccharides) (such as monosaccharides (glucose, etc.), disaccharides), antioxidants (such as SOD, vitamin E, glutathione, at least one compound from the group consisting of polyphenols), hydrophilic polymers (eg polyvinylpyrrolidone), chelating agents (eg EDTA), sugar alcohols (eg mannitol, sorbitol) and glycerol.

In one form, the liquid composition of the present invention may be free of cryoprotectants. As a cryoprotectant, DMSO, glycerin, ethylene glycol, 1,3-propanediol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran sugar, albumin, etc. In one form, the liquid composition of the present invention does not contain a compound selected from the group consisting of DMSO, glycerin, ethylene glycol, 1,3-propanediol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyl At least one compound in the group consisting of ethyl starch, dextran and albumin.

Add deacylated gellan gum or its salts, and acid polysaccharides (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions in the above-mentioned liquid medium, or In the case of its salt, first, use a suitable solvent to deacylate gellan gum or its salt, and maintain a random coil state in a divalent metal cation medium, and the acidity that can be cross-linked by divalent metal ions is more acidic. Sugars (eg, alginic acid) or salts thereof are dissolved or dispersed (as a medium additive). After that, in order to make the deacylated gellan gum or its salt in the liquid composition, and maintain the random coil state in the divalent metal cation medium, and can be cross-linked by the divalent metal ions of acidic polysaccharides (such as The final concentration of alginic acid) or its salt becomes the concentration described in detail above, and the medium additive can be added to the liquid medium. A medium supplement comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions can be prepared separately. ) or a medium supplement of a salt thereof, which are separately added to a liquid medium; can also be prepared comprising deacylated gellan gum or a salt thereof, and maintained in a random coil state in a divalent metal cation medium, and can be obtained by A medium supplement for both acidic polysaccharides (eg, alginic acid) or salts thereof cross-linked by divalent metal ions (ie, deacylated gellan gum or its salts, and maintenance of random coil state in a divalent metal cation medium) , and acidic polysaccharides (such as alginic acid or a mixture of salts thereof) that can be cross-linked by divalent metal ions) are added to the liquid medium. It is preferable to prepare an acidic polysaccharide (such as alginic acid) or its salt that contains deacylated gellan gum or its salt, and maintains a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions. Media supplements for both salts (i.e., deacylated gellan gum or its salts, and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions (e.g., seaweed) acid) or a mixture of its salts), added to the liquid medium.

Here, examples of suitable solvents for preparing medium supplements include aqueous solvents such as water, physiological saline, and PBS, dimethylsulfoxide (DMSO), methanol, ethanol, butanol, propanol, and glycerol. Hydrophilic solvents such as various alcohols such as propylene glycol and butylene glycol are not limited to these. In this case, it is desirable to deacylate gellan gum or its salts in the medium additive, and acidic polysaccharides ( For example, the concentration of alginic acid) or its salt is set to, for example, about 10 to 500 times, preferably about 25 to 100 times, the final concentration in the liquid composition of the present invention as detailed above.

If necessary, deacylation gellan gum or its salts, and acidic polysaccharides (such as alginic acid) or its salts that can maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions Sterilize. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization (autoclave sterilization), filter sterilization, and the like. The material of the filter part when performing filter sterilization (hereinafter sometimes referred to as filter sterilization) is not particularly limited, for example, glass fiber, nylon, PES (polyether sintered), hydrophilic PVDF (polyvinylidene vinyl fluoride), cellulose mixed ester, cellulose acetate, polytetrafluoroethylene, etc. The size of the pores of the filter is not particularly limited, but is preferably 0.1 μm to 10 μm, more preferably 0.1 μm to 1 μm, and most preferably 0.1 μm to 0.5 μm. These sterilization treatments can maintain a random coil state in deacylated gellan gum or its salts, and in a divalent metal cation medium, and can be cross-linked by divalent metal ions. Acidic polysaccharides (such as alginic acid) or The salt can be carried out in the state of solid, and can also be carried out in the state of solution.

The temperature in the high-pressure steam sterilization treatment is usually 105 to 135°C, preferably 115 to 130°C, more preferably 118 to 123°C (for example, 121±1°C). The pressure during the sterilization treatment is usually 0.12-0.32 MPa, preferably 0.17-0.27 MPa, more preferably 0.19-0.23 MPa (for example, 0.21±0.1 MPa). The sterilization treatment time is usually 1 to 60 minutes, preferably 5 to 45 minutes, and more preferably 15 to 25 minutes (for example, 20±1 minutes).

Combination of autoclaving conditions For example, 105-135°C, 0.12-0.32 MPa, 1-60 minutes; Preferably, the temperature is 115℃～130℃, 0.17～0.27 MPa, 5～45 minutes; More preferably, it is 118-123 degreeC (for example, 121±1 degreeC), 0.19-0.23 MPa (for example, 0.21±0.1 MPa), and 15-25 minutes (for example, 20±1 minute).

Acidic polysaccharides (such as alginic acid) that can be cross-linked by divalent metal ions by adding deacylated gellan gum or its salts in a liquid medium and maintaining a random coil state in a divalent metal cation medium or a solution or dispersion of its salt, and in a liquid medium, deacylated gellan gum or its salt, maintained in a random coil state in a divalent metal cation medium, and can be cross-linked by divalent metal ions. Acidic polysaccharides (such as alginic acid) or salts thereof are assembled through metal cations (such as divalent metal cations such as calcium ions) to form a three-dimensional network structure (unshaped structure) to obtain the liquid composition of the present invention. The medium usually contains acidic polysaccharides (such as alginic acid) that are sufficient to dearylate gellan gum or its salts, maintain a random coil state in a divalent metal cation medium, and can be cross-linked by divalent metal ions, or The concentration of metal cations (such as calcium ions) at the concentration of its salts to form a three-dimensional network structure (amorphous structure), therefore, only add deacylated gellan gum or its salts in the liquid medium, and in the divalent metal cation medium The liquid composition of the present invention can be obtained from a solution or dispersion of acidic polysaccharides (eg, alginic acid) or its salts that maintain a random coil state and can be cross-linked by divalent metal ions. Alternatively, it can also be used in the medium additives of the present invention (deacylated gellan gum or its salts, and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions (such as alginic acid) or its salt solution or dispersion) add the medium. Furthermore, deacylated gellan gum or its salts, and acidic polysaccharides (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions can also be used. The liquid composition of the present invention is prepared by mixing salt and medium components (powder medium or concentrated medium) in an aqueous solvent (for example, water containing ion-exchanged water, ultrapure water, or the like). Examples of the mixed form include: (1) mixing a liquid medium with a medium additive (solution); (2) adding deacylated gellan gum or a salt thereof to the liquid medium, and maintaining it in a divalent metal cation medium Solid (powder, etc.) of acidic polysaccharides (such as alginic acid) or its salts in the state of random coils and can be cross-linked by divalent metal ions; (3) Mixing powder medium in medium additive (solution); (4) ) powder medium, and deacylated gellan gum or its salt, and acidic polysaccharides (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions, or The solid (powder, etc.) of its salt is mixed with an aqueous solvent, etc.; however, it is not limited to these. In order to prevent deacylation of gellan gum or its salts in the liquid composition, and acid polysaccharides (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions The distribution of the salt thereof is not uniform, and the form (1) is preferred.

In order to make deacylated gellan gum or its salt, and maintain a random coil state in a divalent metal cation medium, acidic polysaccharides (such as alginic acid) or its salt that can be cross-linked by divalent metal ions are dissolved in Solvents (such as water, liquid medium and other aqueous solvents), or acids that can deacylate gellan gum or its salts and maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions When a polysaccharide (for example, alginic acid) or a salt thereof, and a powder medium are dissolved in a solvent, the mixed solution may be heated in order to promote dissolution. As the heating temperature, for example, 80°C to 130°C can be mentioned, and 100°C to 125°C (for example, 121°C) such as heat sterilization is preferable. After heating, the obtained deacylated gellan gum or its salt, and acid polysaccharides (such as alginic acid) that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions The solution of its salt was cooled to room temperature. By adding the above-mentioned metal cations (for example, divalent metal cations such as calcium ions) to the solution (for example, by adding the solution to a liquid medium), the deacylated gellan gum or its salts are combined with the divalent cations. Acidic polysaccharides (such as alginic acid) or their salts that maintain a random coil state in a metal cation medium and can be cross-linked by divalent metal ions are assembled by metal cations (such as divalent metal cations such as calcium ions), thereby forming Three-dimensional network structure (unshaped structure), the liquid composition of the present invention can be obtained. Or by acid polysaccharide (such as alginic acid) or its acid polysaccharide (such as alginic acid) that maintains a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions in deacylated gellan gum or its salt. When the salt is dissolved in a solvent (such as water, an aqueous solvent such as a liquid medium) containing the above-mentioned metal cations (such as divalent metal cations such as calcium ions), it is heated (such as 80°C to 130°C, preferably 100°C to 125°C ( For example, at 121°C)), the obtained solution is cooled to room temperature, and the deacylated gellan gum or its salt can also be maintained in a random coil state in a divalent metal cation medium, and can be passed through divalent Metal ion-crosslinked acidic polysaccharides (eg, alginic acid) or salts thereof are assembled through metal cations (eg, divalent metal cations such as calcium ions), thereby forming a three-dimensional network structure (amorphous structure).

Furthermore, deacylated gellan gum or its salts have relatively linear structural units. Therefore, when added to a solvent (such as water), a plurality of sugar chains are bundled, so they are not easily dissolved. Among them, acidic polysaccharides (such as alginic acid) or their salts, which are added to the divalent metal cation medium to maintain the state of random coils and can be cross-linked by divalent metal ions, are prepared by L- The two uronic acids of glucuronic acid and β1-4-bonded D-mannuronic acid have a relatively bulky structure, so that the deacylation gellan gum or its salt is inhibited from being bundled, and it becomes relatively easy to dissolve. Therefore, deacylated gellan gum or its salts, and acidic polysaccharides (such as alginic acid) or its salts that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions do not require heating , that is, it can be dissolved in a solvent (for example, an aqueous solvent such as water, liquid medium, etc.) at a relatively low temperature (for example, 0 to 37° C., preferably 10 to 30° C.).

Although the manufacturing method of the liquid composition of this invention is illustrated, it is not limited to this.

Add deacylated gellan gum or its salts, and acidic polysaccharides (such as alginic acid) or its salts that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions Exchange water or ultrapure water. Furthermore, acid polysaccharides (such as alginic acid) that can maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions can be used for deacylation gellan gum or its salts. Stir at a temperature at which the salt dissolves (for example, 5 to 60°C, preferably 5 to 40°C, and more preferably 10 to 30°C) to dissolve until it becomes a transparent state.

After dissolving, if necessary, it is cooled naturally while stirring, and sterilization is performed (for example, autoclave sterilization at 121° C. for 20 minutes, filter filtration). While stirring (for example, a homogenizer, etc.) any medium, the above-mentioned sterilized aqueous solution is added to the medium, and the medium is uniformly mixed. The mixing method of the aqueous solution and the culture medium is not particularly limited, and examples thereof include manual mixing such as pipetting, mixing using machines such as a magnetic stirrer, a mechanical stirrer, a homogenizer, and a homogenizer.

In order to uniformly dearylate gellan gum or its salts, and maintain a random coil state in a divalent metal cation medium, acidic polysaccharides (such as alginic acid) or its salts that can be cross-linked by divalent metal ions Disperse in the liquid medium, for example, add liquid medium in a conical tube, maintain the stirring state with a vortex stirrer, etc., and quickly inject deacylated gellan gum or its salt into the liquid medium from a syringe equipped with an injection needle, and An aqueous solution of acidic polysaccharides (such as alginic acid) or its salts that maintain a random coil state in a divalent metal cation medium and can be cross-linked by divalent metal ions. By using a medium preparation kit (Nissan Chemical FCeM ^TM series preparation kit), it is easy to prepare uniformly dispersed deacylated gellan gum or its salt, and maintain random coils in a divalent metal cation medium Three-dimensional network structure (amorphous structure) formed by the aggregation of acidic polysaccharides (such as alginic acid) or their salts through metal cations (such as divalent metal cations such as calcium ions) that can be cross-linked by divalent metal ions The liquid composition of the present invention.

After mixing, the liquid composition of the present invention can also be filtered using a filter. The size of the pores of the filter used for the filtration treatment is 5 μm to 100 μm, preferably 5 μm to 70 μm, and more preferably 10 μm to 70 μm.

In this specification, the so-called "cellular function" refers to the function possessed by cells. Examples of cell functions include, but are not limited to, proliferative potential, adhesion potential, differentiation potential, maintenance of an undifferentiated state, control of a differentiated state, and ability to secrete secreted factors.

In one form, the liquid composition of the present invention can be applied to mesenchymal stem cells. Mesenchymal stem cells are known to have various functions, and by culturing mesenchymal stem cells in the liquid composition of the present invention, the undifferentiated state, chemotaxis, and secretion of secretory factors of mesenchymal stem cells can be promoted.

In the present specification, "promoting the undifferentiated state of mesenchymal stem cells" means a state in which the mesenchymal stem cells are transformed from the undifferentiated state to a better state and the differentiation of the cells is inhibited. More specifically, for example, it means that the transcription level of undifferentiated markers such as OCT4 or NANOG gene in mesenchymal stem cells is higher than that before the application of the liquid composition of the present invention. In one aspect of the present invention, the transcription level of the OCT4 gene or the NANOG gene is used as an indicator for promoting the undifferentiated state of the mesenchymal stem cells. In this case, the increase rate of the transcription level of the OCT4 gene or the NANOG gene is usually 10%. more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 110%, more than 120%, More than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, more than 200%, more than 210%, more than 220%, more than 230%, more than 240%, more than 250% above, 260% or more, 270% or more, 280% or more, 290% or more, or 300% or more, but not limited to these.

In addition, by culturing or storing mesenchymal stem cells in the liquid composition of the present invention, the chemotaxis of mesenchymal stem cells can be promoted. The promotion of chemotaxis of mesenchymal stem cells can be confirmed by measuring the increase of chemotaxis-related factors of mesenchymal stem cells by a known method. Examples of such factors include, but are not limited to, the transcription levels of the CXCR4 gene, the MMP2 gene, the VCAM-1 gene, the integrin α4 gene, and the integrin β1 gene.

Furthermore, by culturing the mesenchymal stem cells in the liquid composition of the present invention, the secretion amount of the factors secreted by the mesenchymal stem cells can be promoted. In the liquid composition of the present invention, "promoting the secretion of secreted factors of mesenchymal stem cells" means to increase the production capacity of secreted factors of mesenchymal stem cells and to increase the secretion of the factors to the outside of the cells. Furthermore, in the present specification, it means that the production and secretion of the secreted factor are at least increased compared with the secreted amount of the specific secreted factor as a control with respect to the increased amount of the secreted factor by applying the liquid composition of the present invention. 120% or more, at least 130% or more, at least 140% or more, at least 150% or more, at least 160% or more, at least 170% or more, at least 180% or more, at least 190% or more, at least 200% or more, at least 300% or more, At least 400% or more, at least 500% or more, at least 600% or more, at least 700% or more, at least 800% or more, at least 900% or more, at least 1000% or more. For the measurement of the secreted factor, for example, when the factor is a protein, a known method such as an ELISA (enzyme-linked immunosorbent assay) method or a flow cytometry method can be used.

Examples of the secreted factor include TSG-6 (TNF-stimulated gene 6 protein, tumor necrosis factor-stimulated gene 6 protein), STC-1 (Stanniocalcin-1, Stanniocalcin-1), ANG (Angiogenin, angiopoietin), EGF (Epidermal Growth Factor, epidermal growth factor), MCP-1 (Monocyte Chemotactic Protein-1, monocyte chemoattractant protein-1), ENA-78 (epithelial-derived neutrophil-activating peptide 78, epithelial cell-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor, basic fibroblast growth factor), IL-6 (Interleukin-6, interleukin-6), IL-8 (Interleukin -8, interleukin-8), VEGF (Vascular endothelial growth factor, vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D, vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase, Matrix metalloproteinase tissue inhibitor), PDGF (Platelet-Derived Growth Factor, platelet-derived growth factor), TGF-β (transforming growth factor-β, transforming growth factor-β), HGF (Hepatocyte growth factor, hepatocyte proliferation factor) and PGE2 (Prostaglandin E2, prostaglandin E2), etc., but not limited to these.

In another form, the medium composition of the present invention can be applied to hepatocytes. By applying the medium composition of the present invention to hepatocytes, the cell adhesion ability of the cells can be promoted. In this regard, the liquid composition of the present invention can be preferably used for preparing hepatocytes for transplantation.

2. Method for promoting cell function Further, the present invention provides a method for promoting cell function (hereinafter sometimes referred to as "the method of the present invention"), which comprises culturing cells in the liquid composition of the present invention.

The method of the present invention is accomplished by culturing or storing cells in the above-mentioned liquid composition of the present invention. The liquid composition, cell species, etc. in the method of the present invention are the same as those described in "1. Liquid composition".

In one form of the method of the present invention, the culture of cells can be performed by suspension culture (preferably suspension culture).

In a preferred form of the present invention, the cells are seeded and cultured in the state of single cells. In other words, in a preferred form of the present invention, the cells can be cultured without forming a sphere.

Hereinafter, the examples of the liquid composition of the present invention will be specifically described to explain the present invention in more detail, but the present invention is not limited to these examples. [Example]

[Test Example 1] Preparation of polysaccharide mixed aqueous solution 0.67 parts by mass of sodium alginate (ALG) (Kimica Algin IL-2, manufactured by KIMICA Co., Ltd.) and 0.33 parts by mass of deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sanjing Co., Ltd.) ) was added to a glass culture bottle containing 99 parts by mass of purified water, dissolved by stirring, and then passed through a 0.22 μm sterile filter (Corning Filtration System 430767, manufactured by Corning Corporation) to prepare 1 mass % concentration of polysaccharide mixed aqueous solution.

[Test Example 2] Preparation of ALG/DAG preparation medium composition The medium composition was prepared using a medium preparation kit (Nissan Chemical FCeM (registered trademark) series preparation kit). Human room prepared by adding MSC Nutristem (registered trademark) XF Basal Medium (05-200-1A, manufactured by Biological Industries) to MSC Nutristem (registered trademark) XF Supplement Mix (05-200-1U, manufactured by Biological Industries). 49.5 mL of Xeno-Free medium for leaf stem cells was dispensed into the 50 mL conical tube attached to the above-mentioned kit, and an adapter as a component of the kit was installed. Insert the front end of the disposable syringe filled with 0.5 mL of the polysaccharide mixture obtained in Test Example 1 into the cylindrical part of the adapter and connect it, manually press the plunger of the syringe, and quickly inject the polysaccharide mixture in the syringe into the container. The liquid medium composition with a final polysaccharide concentration of 0.01% (w/v) (hereinafter referred to as ALG/DAG preparation medium composition) was prepared by instantaneously mixing it with the medium.

[Test Example 3] Gene expression analysis of human interumbilical cord-derived leaf stem cells statically cultured in a three-dimensional suspension state in the ALG/DAG compounded medium composition The ALG/DAG compounded medium composition prepared in Test Example 2 was analyzed for gene expression , and inoculated with human interumbilical cord-derived stem cells (C-12971, manufactured by TAKARA BIO INC.) at 100,000 cells/mL, added to a 1.5 mL tube, and kept at room temperature for 6 days. 350 μL of RLT solution (RNeasy mini kit (manufactured by QIAGEN, #74106)) was added to the human interumbilical cord-derived stem cells used for inoculation to obtain an RNA extraction solution in advance. After 6 days, transfer from the 1.5 mL tube to the 15 mL tube, and add chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) to 20% (v/v) After 5 minutes of centrifugation at 300×g, the culture supernatant was removed. Next, 350 μL of an RLT solution (RNeasy mini-extraction kit (manufactured by QIAGEN, #74106)) was added, and this was used as an RNA extraction solution. Also, as a comparison object, an RNA extraction solution was obtained from human interumbilical cord-derived leaf stem cells after two-dimensional culture in a 10 cm culture dish for 3 days at 37°C in the presence of 5% CO ₂ . Add 350 μL of 70% ethanol to the RNA extraction solution, add it to the RNeasy spin column, and centrifuge at 8000×g for 15 seconds. Then, 700 μL of RW1 solution was added to the RNeasy spin column, and centrifugation was performed at 8000×g for 15 seconds. Next, 500 μL of RPE solution was added, and centrifugation was performed at 8000×g for 15 seconds. Furthermore, 500 μL of the RPE solution was added, followed by centrifugation at 8000×g for 2 minutes. An RNase-free solution is added to the RNA present in the RNeasy spin column to dissolve it. Next, cDNA was synthesized from the obtained RNA using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by TAKARA BIO INC., #RR037A). Real-time PCR was performed using the synthesized cDNA, Premix EX Taq (all real-time) (manufactured by TAKARA BIO INC., #RR039A), and Taq man probe (Taq man Probe) (manufactured by Applied Bio Systems). As the Taq man probe (manufactured by Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs04399610_g1 for NANOG, Hs00607978_s1 for CXCR4, and Hs99999905_m1 for GAPDH. The machine uses a real-time PCR7500. In the analysis, the relative value obtained by correcting the value of each target gene with the value of GAPDH was calculated and compared.

As a result, compared with the human interumbilical cord-derived stem cells on day 0 or two-dimensionally cultured, the human interumbilical cord-derived stem cells stored in the ALG/DAG medium composition in suspension and storage The relative expression levels of OCT4, NANOG gene or targeting-related CXCR4 gene that showed undifferentiated nature increased. The relative values of the expression of each gene are shown in Table 1.

[Table 1] OCT4 NANOG CXCR4 Day 0 0.54 0.64 0.72 Day 6 1.72 1.43 1.97 2D 0.56 0.69 0.56

[Test Example 4] The amount of production of TSG-6 derived from human interumbilical cord leaf stem cells stored in a three-dimensional suspended state in the ALG/DAG-prepared medium composition was in the ALG/DAG-prepared medium composition prepared in Test Example 2 The medium was seeded with human interumbilical cord-derived stem cells (C-12971, manufactured by TAKARA BIO INC.) at 100,000 cells/ml, added to a 1.5 mL tube, and allowed to stand at room temperature for 7 days. After 6 days, transfer from the 1.5 mL tube to the 15 mL tube, and add chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) to 20% (v/v) After 5 minutes of centrifugation at 300×g, the culture supernatant was removed. After removing the culture supernatant, add Xeno-Free medium for human mesenchymal stem cells (sample 1) or TNF containing a final concentration of 20 ng/mL in a 15 mL tube so as to reach 60,000 cells/mL. -α (#210-TA, manufactured by R&D Systems, Inc.) in Xeno-Free medium for human mesenchymal stem cells (Sample 2), added to a 24-well flat-bottom ultra-low adhesion surface microplate (#3473, Corning company) in the hole. In addition, as a comparison object, human mesenchymal stem cells were inoculated with human mesenchymal stem cells at 60,000 cells/mL after two-dimensional culture using a 10 cm culture dish at 37°C in the presence of 5% CO ₂ . Xeno-free (Xeno-Free) medium (Sample 3) or human mesenchymal stem cells containing a final concentration of 20 ng/mL TNF-α (#210-TA, manufactured by R&D Systems) -Free) medium (sample 4), added to a 24-well flat-bottom ultra-low adhesion surface microplate. The culture plate was cultured in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ) for 1 day. On the first day, the cells and medium were transferred from each well to a 15 mL tube, centrifuged at 300×g for 3 minutes, and then 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 collected. , the cells were suspended, and after standing at room temperature for 10 minutes, the luminescence intensity (RLU value) was measured using Enspire (manufactured by Perkin Elmer), and the luminescence value in the medium alone was subtracted to determine the number of viable cells. Next, TSG-6 contained in the recovered culture supernatant was quantified using ELISA. To Maxisorp flat bottom (#44-2404-21, manufactured by Thermo Fisher Scientific), 50 μL/well of TSG-6 antibody diluted to 10 μg/mL with 0.2M carbonic acid-bicarbonate buffer (pH 9.2) was added (#sc-65886, manufactured by Santacruz Corporation), and left to stand at 4°C for 24 hours. After 24 hours, 300 μL of Tween-20 (#P7949) was added to D-PBS(-) (#043-29791, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 0.05% (v/v). , manufactured by Sigma-Aldrich Co., Ltd.) (hereinafter referred to as PBST solution), and then removed. Repeat this operation 3 times. 100 μL of a PBST solution containing 5% BSA (#A2153, manufactured by Sigma-Aldrich) was added, and the solution was allowed to stand at room temperature for 30 minutes. After the solution was discarded, 300 μL of PBST solution was added and removed. Repeat this operation 3 times. Next, the prepared TSG-6 for calibration curve (#2104-TS, manufactured by R&D Systems) and 50 μL of the evaluation sample were added to each well, and the mixture was left to stand at room temperature for 2 hours. After the solution was discarded, 300 μL of PBST solution was added and removed. Repeat this operation 3 times. 50 μL of a solution of biotin-labeled anti-human TSG-6 antibody (#BAF2104, manufactured by R&D Systems) diluted to 5 μg/mL with PBST was added, and the solution was allowed to stand at room temperature for 120 minutes. After the solution was discarded, 300 μL of PBST solution was added and removed. Repeat this operation 3 times. 50 μL of a solution of Streptavidin-HRP (#ab7403, manufactured by Abcam) diluted to 200 ng/mL with a PBST solution was added, and the solution was allowed to stand at room temperature for 30 minutes. After the solution was discarded, 300 μL of PBST solution was added and removed. Repeat this operation 3 times. 100 μL of substrate solution (#52-00-03, manufactured by KPL) was added, and the mixture was left to stand at room temperature for 15 minutes. Finally, 100 μL of stop solution (#50-85-06, manufactured by KPL) was added, and the absorbance at 450 nm was measured. The concentration of TSG-6 contained in each sample was calculated by 4-parameter logarithmic regression of the calibration curve. In order to calculate the average secretion per cell, the relative value obtained by dividing the calculated amount of TSG-6 by the luminescence intensity was calculated.

As a result, compared with two-dimensionally cultured human interumbilical cord-derived stem cells, the human interumbilical cord-derived stem cells cultured in static suspension in the ALG/DAG formulation medium composition were treated with TNF-α. The average production of TSG-6 per cell was higher. The relative value of each sample is shown in Table 2.

[Table 2] Cultivation method TNFα relative value Sample 1 Method of the present invention - 5.07E-06 Sample 2 Method of the present invention + 7.06E-05 Sample 3 2D culture - 5.44E-06 Sample 4 2D culture + 1.28E-05

[Test Example 5] Promotion of E-cadherin adhesion of primary cynomolgus monkey-derived hepatocytes cultured in a three-dimensional suspension state in an ALG/DAG-formulated medium composition

Preparation of culture dishes coated with E-cadherin peptide E-cadherin peptide (MAPTrix (registered trademark)-E, manufactured by Kollodis BIOSCIENCES, #167021, #167061) was diluted with sterilized water to 0.2 mg/mL, respectively, and placed in a 96-well cell culture plate (manufactured by Corning, # 3585) was added with 100 μL and stored under refrigeration for one day and night. Only sterilized water was added to the wells without peptides. The peptide solution was removed before use, washed with D-PBS(-), and used.

In the culture method of the present invention and the control culture method, 2 tubes (1 million to 5 million per tube) of frozen primary cynomolgus monkey hepatocytes (manufactured by Ina Research Co., Ltd.) were mixed and suspended in HBM medium. Dispense equally into 2 tubes. Thereafter, centrifugation was performed at 50×g for 3 minutes, and the cell pellet in one tube was suspended in 2.5 ml of HBM medium (control culture method). In the control culture method, 100 μL/well was inoculated into a 96-well cell culture plate (manufactured by Corning, #3585) and a culture plate coated with each E-cadherin peptide, and the culture was continued at 37°C and 5% CO ₂ . 2 hours.

The cell pellet in another tube was suspended in 5 ml of ALG/DAG preparation medium composition (the culture method of the present invention), and 5 mL/well was inoculated into a 6-well ultra-low adhesion culture plate (manufactured by Corning Company, #3471), A 2-hour pre-incubation was performed at 37°C, 5% CO ₂ . After 2 hours, transfer from the well to a 15 mL tube, and add a mixture of chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) to 10% (v/v) Aqueous solution), the remaining cells in the wells were further recovered with D-PBS(-), and finally 5 times the amount of D-PBS(-) was added, followed by centrifugation at 50 × g for 3 minutes, and the culture supernatant was removed. The precultured cell pellets were suspended in 2.5 ml of HBM medium, and 100 μL/well were inoculated into a 96-well cell culture plate (manufactured by Corning, #3585) and a culture plate coated with each E-cadherin peptide. The cells were further cultured for 2 hours under 5% CO ₂ .

Determination of the total number of cells inoculated In the control culture method, for the culture solution 2 hours after inoculation, in the culture method of the present invention, in the culture method of the present invention, 100 μL of ATP reagent (CellTiter- Glo (registered trademark) cell viability assay by luminescence method, manufactured by Promega Corporation), suspended cells at room temperature for about 10 minutes, and then measured the luminescence intensity (RLU value) using FlexStation3 (manufactured by Molecular Devices Corporation), minus only The luminescence value of the medium was used to determine the number of viable cells.

Determination of the number of adherent cells The culture supernatant was removed from the 96-well cell culture dish (not treated with E-cadherin peptide) and the inoculation wells in the culture dish coated with each E-cadherin peptide, added 200 μL of HBM medium, removed the medium, and repeated After this washing step was performed twice, 100 μL of HBM medium and 100 μL of ATP reagent were added to suspend the remaining cells due to adhesion. After standing at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured by FlexStation 3, and the value was decreased. The number of adherent cells was determined by the luminescence value when only the medium was removed. The adhesion rate was calculated by dividing the number of adherent cells (RLU value) under each condition by the total number of cells (RLU value) in each culture method.

As a result, by pre-culturing in the ALG/DAG-prepared medium composition, a marked effect of improving the adhesion of primary cynomolgus monkey hepatocytes to the culture plate coated with E-cadherin peptide was confirmed. The results of the adhesion rate are shown in Table 3.

[table 3] Adhesion rate (%; adhesion RLU/total cell number RLU) not processed Cat. No. 167021 Cat. No. 167061 control culture 2.2 6.3 7.1 The culture method of the present invention 4.8 33.1 40.5

[Test Example 6] Promotion of Adhesion to E-cadherin of Primary Human-derived Hepatocytes Cultured in Three-Dimensional Suspension in ALG/DAG-Formulated Medium Composition

Preparation of culture dishes coated with E-cadherin peptide E-cadherin peptide (MAPTrix (registered trademark)-E, manufactured by Kollodis BIOSCIENCES, #167021) was diluted with sterilized water to 0.2 mg/mL, respectively, and placed in a 96-well cell culture dish (manufactured by Corning, #3585). Add 100 μL and keep under refrigeration for one day and night. Only sterilized water was added to the wells without peptides. The peptide solution was removed before use, washed with D-PBS(-), and used.

In the culture method of the present invention and the control culture method, one tube (about 5 million cells/tube) of frozen primary human hepatocytes (manufactured by Xeno Tech) was mixed, suspended in HBM medium, and dispensed equally into two tubes. middle. Thereafter, centrifugation was performed at 50×g for 3 minutes, and the cell pellet in one tube was suspended in 2.5 ml of HBM medium (control culture method). In the control culture method, 100 μL/well was inoculated into a 96-well cell culture plate (manufactured by Corning, #3585), a culture plate coated with E-cadherin peptide, and a type I collagen coated plate. The culture plate (manufactured by IWAKI, #4860-010) was further cultured at 37° C., 5% CO ₂ for 2 hours.

The cell pellet in another tube was suspended in 5 ml of ALG/DAG preparation medium composition (the culture method of the present invention), and 5 mL/well was inoculated into a 6-well ultra-low adhesion culture plate (manufactured by Corning Company, #3471), A 2-hour pre-incubation was performed at 37°C, 5% CO ₂ . After 2 hours, transfer from the well to a 15 mL tube, and add a mixture of chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) to 10% (v/v) Aqueous solution), the remaining cells in the wells were further recovered with D-PBS(-), and finally 5 times the amount of D-PBS(-) was added, followed by centrifugation at 50 × g for 3 minutes, and the culture supernatant was removed. The pre-cultured cell pellets were suspended in 2.5 ml of HBM medium, and 100 μL/well were inoculated into a 96-well culture plate (manufactured by Corning, #3585), a culture plate coated with E-cadherin peptide and coated with type I collagen A protein culture plate (manufactured by IWAKI, #4860-010) was additionally cultured at 37°C under 5% CO ₂ for 2 hours.

Determination of the total number of cells inoculated. In the control culture method, for the culture solution inoculated for 2 hours, in the culture method of the present invention, in the culture method of the present invention, for the culture solution inoculated for 2 hours after pre-culture in the ALG/DAG preparation medium composition, add 100 μL of ATP reagent ( CellTiter-Glo ^™ Luminescence Assay for Cell Viability, manufactured by Promega), suspended cells and allowed to stand at room temperature for about 10 minutes, then measured the luminescence intensity (RLU value) using FlexStation3 (manufactured by Molecular Devices), minus the medium alone The luminescence value at the time was used to measure the number of living cells.

Determination of the number of adherent cells The culture supernatant was removed from the inoculation wells in the 96-well plate (untreated with E-cadherin peptide), the plate coated with E-cadherin peptide, and the plate coated with collagen type I, and added 200 μL of HBM medium, remove the medium, repeat this washing step twice, add 100 μL of HBM medium and 100 μL of ATP reagent to suspend the remaining cells due to adhesion, and let stand at room temperature for about 10 minutes, then use FlexStation3 The number of adherent cells was determined by measuring the luminescence intensity (RLU value) and subtracting the luminescence value in the medium only. The adhesion rate was calculated by dividing the number of adherent cells (RLU value) under each condition by the total number of cells (RLU value) in each culture method.

As a result, by pre-culturing in the ALG/DAG-prepared medium composition, it was confirmed that significant human primary hepatocytes migrated to the E-cadherin peptide-coated culture plate or the type-1 collagen-coated culture plate. Adhesion improvement effect. The results of the adhesion rate to E-cadherin peptide are shown in Table 4, and the culture plate coated with type 1 collagen is shown in Table 5.

[Table 4] Adhesion rate (%; adhesion RLU/total number of cells RLU×100) not processed E-Cadherin Peptide (#167021) control culture 1 3.3 The culture method of the present invention 2.5 17.0

[table 5] Adhesion rate (%; adhesion RLU/total number of cells RLU×100) type 1 collagen control culture 24.2 The culture method of the present invention 66.1

[Test Example 7] Preparation of ALG/DAG-mixed medium composition A medium composition was prepared using a medium preparation kit (Nissan Chemical FceM (registered trademark) series preparation kit). 49.2 mL of StemFit (registered trademark) mesenchymal stem cell medium (manufactured by Ajinomoto Co., Ltd.) was dispensed into a 50 mL conical tube attached to the above-mentioned set, and an adapter, which is a component of the set, was attached. Insert the front end of the disposable syringe filled with 0.8 mL of the polysaccharide mixture obtained in Test Example 1 into the cylindrical part of the adapter and connect it, manually press the plunger of the syringe, and quickly inject the polysaccharide mixture in the syringe into the container. The liquid medium composition with a final polysaccharide concentration of 0.016% (w/v) (hereinafter referred to as ALG/DAG preparation medium composition) was prepared by instantaneously mixing it with the medium.

[Test Example 8] Analysis of the expression of surface markers of human interumbilical cord-derived leaf stem cells cultured in a three-dimensional suspension state in an ALG/DAG-prepared medium composition The leaf stem cells derived from human umbilical cord (C-12971, manufactured by TAKARA BIO INC.) were suspended at 100,000 cells/mL in the ALG/DAG preparation medium composition prepared in Test Example 7 (final concentration: 0.016%), 2 mL was inoculated into a Stemfull (registered trademark) 15 mL centrifuge tube (MS-90150, manufactured by SUMITOMO BAKELITE). After the inoculation, the centrifuge tube was sealed and kept at room temperature for 7 days. After 7 days, add chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) to the cell suspension in a 15 mL centrifuge tube in a manner of 20% (v/v). After 5 minutes of centrifugation at 300×g, the culture supernatant was removed. After washing the obtained cells with SM buffer (2% FBS/PBS), BV650 mouse anti-human CD105 (563466, manufactured by BD Biosciences), BV421 mouse anti-human CD73 (562430, manufactured by BD Biosciences), and APC were added, respectively. Mouse anti-human CD90 (559869, manufactured by BD Biosciences), PE mouse anti-human CD34 (555822, manufactured by BD Biosciences), FITC anti-CD11b antibody [M1/70] (ab24874, manufactured by Abcam), and incubated at room temperature in the dark 30 minutes. As negative controls, BV650 mouse IgG1, k isotype control (563231, manufactured by BD Biosciences), BV421 mouse IgG1, k isotype control (562438, manufactured by BD Biosciences), and APC mouse IgG1, kappa isotype control (555751) were used, respectively. , manufactured by BD Biosciences), PE mouse IgG1, κ isotype control (555749, manufactured by BD Biosciences), FITC rat IgG2b, κ monoclonal [eB149/10H5]-isotype control (ab136125, manufactured by Abcam). The stained cells were washed twice with SM buffer, and then measured by FACSLSR Fortessa X-20 (manufactured by BD Biosciences).

In addition, cells obtained by adding a chelating agent and centrifuging in the same manner as above after culturing in the ALG/DAG medium composition for 7 days were suspended in StemFit (registered trademark) mesenchymal stem cell medium (manufactured by Ajinomoto Co., Ltd.), 1×10 ⁵ cells/well were seeded in a 6-well culture dish, and then re-seeded for flat culture, and cultured at 37° C. in a 5% CO ₂ environment. Three days later, the cells were detached into single cells using a Detach kit (D13101, manufactured by TAKARA BIO INC.), and then the FACS measurement was carried out in the same manner as above.

As a result, the human interumbilical cord-derived leaf line stem cells kept in suspension and stored in the ALG/DAG medium composition for 7 days maintained the expression of the positive marker of MSC, and the increase of the expression of the negative marker was not confirmed, indicating that Maintain the properties of MSC. In addition, the cells cultured by this culture method were re-seeded for flat culture, and as a result, the cells exhibited a normal adherent form, and the expression of MSC markers was maintained without problems. The ratio of positive cells for each marker is shown in Table 6.

[Table 6] Positive rate Day 0 (flat culture) Day 7 (Cultivation method of the present invention) Day 10 (re-seeded for flat culture) positive marker CD73 99.40% 99.90% 99.90% CD90 99.40% 99.80% 99.70% CD105 99.20% 93.80% 98.70% negative marker CD34 0.02% 0.04% 0.00% CD11b 0.14% 0.29% 0.89%

[Test Example 9] Analysis of the expression of surface markers of human interumbilical cord-derived leaf stem cells cultured in a three-dimensional suspension state in an ALG/DAG-prepared medium composition The leaf stem cells derived from human umbilical cord (C-12971, manufactured by TAKARA BIO INC.) were suspended at 100,000 cells/mL in the ALG/DAG preparation medium composition prepared in Test Example 7 (final concentration: 0.016%), 2 mL was inoculated into a Stemfull (registered trademark) 15 mL centrifuge tube (MS-90150, manufactured by SUMITOMO BAKELITE). After the inoculation, the centrifuge tube was sealed and kept at room temperature for 7 days. After 3, 7, 11, and 15 days, add chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% to the cell suspension in a 15 mL centrifuge tube to make it 20% (v/v) % (w/v) in a mixed aqueous solution), centrifuged at 300×g for 5 minutes, and then removed the culture supernatant. The obtained cells were washed with SM buffer (2% FBS/PBS), and BV421 mouse anti-human CD271 (562562, manufactured by BD Biosciences) or BV421 mouse IgG1, k isotype control (562438, manufactured by BD Biosciences) was added, respectively. ) and incubated at room temperature for 30 minutes in the shade. The stained cells were washed twice with SM buffer, and then measured by FACSLSR Fortessa X-20 (manufactured by BD Biosciences).

As a result, the expression of CD271 was not confirmed in the human interumbilical cord-derived stem cells used in this experiment that were cultured on a flat surface, but the human interumbilical cord-derived stem cells stored in suspension in the ALG/DAG medium composition The percentage of positive cells in the stem cells continued to increase until day 7, and the percentage of positive cells was maintained thereafter. The ratio of positive cells for each marker is shown in Table 7.

[Table 7] Positive rate (planar culture) (Cultivation method of the present invention) Day 0 Day 3 Day 7 Day 11 Day 15 CD271 0.27% 11.30% 30.30% 31.00% 29.00%

[Test Example 10] Preparation of additives

[Table 8] content concentration, batch, etc. 1 Blank (sterilized water) Filter Sterilize MiliQ 2 Deacylated Gellan Gum + Oligomeric Alginic Acid 1%, KFN20420 3 Deacylated Gellan Gum + Alginic Acid 1%

[Test Example 12] Preparation of polysaccharide-prepared medium composition The medium composition was prepared using a medium preparation kit (Nissan Chemical FCeM (registered trademark) series preparation kit). 39.4 mL of StemFit (registered trademark) mesenchymal stem cell medium (manufactured by Ajinomoto Co., Ltd.) was dispensed into a 50 mL conical tube attached to the above-mentioned set, and an adapter, which is a component of the set, was attached. Insert the front end of the disposable syringe filled with 0.6 mL of each polysaccharide mixture obtained in Test Example 11 into the cylindrical part of the adapter and connect it, manually press the plunger of the syringe, and quickly eject the polysaccharide mixture in the syringe. The liquid medium composition with a final polysaccharide concentration of 0.015% (w/v) was prepared by pouring it into a container and instantaneously mixing it with the medium.

[Test Example 13] Gene analysis of human interumbilical cord-derived leaf stem cells cultured in a three-dimensional suspension state in a polysaccharide-prepared medium composition The human interumbilical cord-derived leaf stem cells (C-12971, TAKARA BIO INC. company) at 100,000 cells/mL suspended in each medium composition (additive final concentration 0.015%) prepared in Test Example 12, and 2 mL was inoculated into an ultra-low adhesion 6-well culture plate (3471, manufactured by Corning), Let stand at room temperature. After 4 or 7 days, the cell suspension was transferred to a 15 mL centrifuge tube, and a chelating agent (EDTA-2Na 0.033% (w/v) and sodium citrate 0.007% (w/v) was added to make it 20% (v/v). /v) mixed aqueous solution), centrifuged at 300×g for 5 minutes, and then the culture supernatant was removed. Next, 350 μL of an RLT solution (RNeasy mini-extraction kit (manufactured by QIAGEN, #74106)) was added, and this was used as an RNA extraction solution. As a comparison object, stem cells derived from human interumbilical cord leaf lines were suspended in StemFit (registered trademark) mesenchymal stem cell medium, and seeded at 50,000 cells/well on iMatrix-511 silk (892 021, 0.5 μg/cm ² ). A 6-well culture plate manufactured by Matrixome, Inc., was cultured on a flat surface at 37°C in a 5% CO ₂ environment, and on the 4th day of culture, it was subcultured using a Detach kit (D13101, manufactured by TAKARA BIO INC.). cell. On day 4 or 7 of culture, RNA extraction solutions were obtained from these cells. Add 350 μL of 70% ethanol to the RNA extraction solution, add it to the RNeasy spin column, and centrifuge at 8000×g for 15 seconds. Then, 700 μL of RW1 solution was added to the RNeasy spin column, and centrifugation was performed at 8000×g for 15 seconds. Next, 500 μL of RPE solution was added, and centrifugation was performed at 8000×g for 15 seconds. Furthermore, 500 μL of the RPE solution was added, followed by centrifugation at 8000×g for 2 minutes. RNase-free solution is added to the RNA present in the RNeasy spin column to dissolve it. Next, cDNA was synthesized from the obtained RNA using PrimeScript RT Reagent Kit (All Real Time) (manufactured by TAKARA BIO INC., #RR037A). Real-time PCR was performed using the synthesized cDNA, Premix EX Taq (all real-time) (manufactured by TAKARA BIO INC., #RR039A), and Taq man probe (manufactured by Applied Bio Systems). As the Taq man probe (manufactured by Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs04399610_g1 for NANOG, and Hs99999905_m1 for GAPDH. The machine uses a real-time PCR7500. In the analysis, the relative value obtained by correcting the value of each target gene with the value of GAPDH was calculated and compared.

As a result, it was confirmed that the relative expression levels of OCT4 and NANOG genes showing undifferentiated properties were increased in each of the additives evaluated this time. The relative values of OCT4 gene expression are shown in Table 9, and the relative values of NANOG gene expression are shown in Table 10.

[Table 9] flat culture Deacylated Gellan Gum + Oligomeric Alginic Acid Deacylated Gellan Gum + Alginic Acid Day 0 0.21 - - Day 4 0.37 1.41 1.14 Day 7 0.53 1.87 1.92

[Table 10] flat culture Deacylated Gellan Gum + Oligomeric Alginic Acid Deacylated Gellan Gum + Alginic Acid Day 0 0.26 - - Day 4 0.24 1.48 1.37 Day 7 0.48 1.89 1.67 [Industrial Availability]

According to the present invention, for example, the adhesion ability of hepatocytes can be improved, and the function of mesenchymal stem cells, which are reduced due to repeated passage in 2D culture, etc., can be improved. In other words, the present invention can achieve an improvement in the survival rate at the time of hepatocyte transplantation, or quality adjustment or high quality in the mass production of mesenchymal stem cells. Therefore, the present invention is very useful in the field of regenerative medicine.

This application is based on Japanese Patent Application No. 2020-084151 (filing date: May 12, 2020 ) filed in Japan, and the entire contents of which are incorporated in this specification.

Claims (20)

A liquid composition comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked by a divalent metal ion , The liquid composition is used to promote cellular function. The liquid composition of claim 1, wherein the concentration of the deacylated gellan gum or its salt in the liquid composition is 0.002 to 0.1 (w/v)% in terms of the free form of the deacylated gellan gum, The concentration of the acidic polysaccharides or their salts is 0.004 to 0.2 (w/v)% in terms of free form, and the mass ratio of the acidic polysaccharides or their salts relative to deacylated gellan gum or its salts is in terms of free forms. , is 1 or more. The liquid composition of claim 1 or 2, wherein the acidic polysaccharide is any one selected from the group consisting of alginic acid, pectin and pectic acid. The liquid composition of claim 3, wherein the acidic polysaccharide is alginic acid. The liquid composition of any one of claims 1 to 4, which further contains metal cations. The liquid composition of claim 5, wherein the metal cation is calcium ion. The liquid composition according to any one of claims 1 to 6, wherein the cells are mesenchymal stem cells, and the cell function to be promoted is selected from the group consisting of undifferentiated maintenance state, chemotaxis, and ability to secrete secreted factors at least one of them. The liquid composition of claim 7, wherein the cellular function to be promoted is the ability to secrete a factor. The liquid composition of claim 8, wherein the secreted factor is selected from the group consisting of TSG-6 (TNF-stimulated gene 6 protein, tumor necrosis factor-stimulated gene 6 protein), STC-1 (Stanniocalcin-1, Stanniocalcin-1, Stanniocalcitonin -1), ANG (Angiogenin, angiopoietin), EGF (Epidermal Growth Factor, epidermal growth factor), MCP-1 (Monocyte Chemotactic Protein-1, monocyte chemoattractant protein-1), ENA-78 (epithelial- derived neutrophil-activating peptide 78, epithelial cell-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor, basic fibroblast growth factor), IL-6 (Interleukin-6, interleukin-6), IL-8 (Interleukin-8, interleukin-8), VEGF (Vascular endothelial growth factor, vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D, vascular endothelial growth factor-D), TIMP (Tissue At least one of the group consisting of inhibitors of matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor, platelet-derived growth factor) and TGF-β (transforming growth factor-β, transforming growth factor-β) one. The liquid composition according to any one of claims 1 to 6, wherein the cells are hepatocytes, and the cell function to be promoted is cell adhesion ability. A method for promoting cell function, comprising culturing cells in the following liquid composition, the above-mentioned liquid composition comprising deacylated gellan gum or a salt thereof, and maintaining a random coil state in a divalent metal cation medium , and acidic polysaccharides or their salts that can be cross-linked by divalent metal ions. The method of claim 11, wherein the concentration of the deacylated gellan gum or its salt in the liquid composition is 0.002 to 0.1 (w/v)% in terms of the free form of deacylated gellan gum, and the acidic The concentration of polysaccharides or their salts is 0.004 to 0.2 (w/v)% in terms of free form, and the mass ratio of the acidic polysaccharides or their salts relative to deacylated gellan gum or its salts, in terms of free forms, is 1 or more. The method of claim 11 or 12, wherein the acidic polysaccharide is any one selected from the group consisting of alginic acid, pectin and pectic acid. The method of claim 13, wherein the acidic polysaccharide is alginic acid. The method of any one of claims 11 to 14, which further contains a metal cation. The method of claim 15, wherein the metal cation is a calcium ion. The method of any one of claims 11 to 16, wherein the cells are mesenchymal stem cells, and the cell function to be promoted is selected from the group consisting of undifferentiated maintenance state, chemotaxis, and ability to secrete secreted factors at least one. The method of claim 17, wherein the cellular function to be promoted is the ability to secrete a secreted factor. The method of claim 18, wherein the secreted factor is selected from the group consisting of TSG-6 (TNF-stimulated gene 6 protein, tumor necrosis factor-stimulated gene 6 protein), STC-1 (Stanniocalcin-1, Stanniocalcin-1) ), ANG (Angiogenin, angiopoietin), EGF (Epidermal Growth Factor, epidermal growth factor), MCP-1 (Monocyte Chemotactic Protein-1, monocyte chemoattractant protein-1), ENA-78 (epithelial-derived neutrophil -activating peptide 78, epithelial cell-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor, basic fibroblast growth factor), IL-6 (Interleukin-6, interleukin-6), IL- 8 (Interleukin-8, interleukin-8), VEGF (Vascular endothelial growth factor, vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D, vascular endothelial growth factor-D), TIMP (Tissue inhibitors of At least one of the group consisting of matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor, platelet-derived growth factor) and TGF-β (transforming growth factor-β, transforming growth factor-β). . The method of any one of claims 11 to 16, wherein the cells are hepatocytes, and the cell function to be promoted is cell adhesion ability.