WO2021230274A1

WO2021230274A1 - Liquid composition for functional enhancement of cells

Info

Publication number: WO2021230274A1
Application number: PCT/JP2021/017995
Authority: WO
Inventors: 克彦木田; 達朗金木; 大輔畑中; 寿人林; 志保阿武
Original assignee: 日産化学株式会社
Priority date: 2020-05-12
Filing date: 2021-05-12
Publication date: 2021-11-18
Also published as: TW202208614A; JPWO2021230274A1

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, which improves cell function by suspending and statically culturing cells in a liquid composition, and a method for promoting cell function using the same.

In the field of regenerative medicine, the construction of organ regeneration means using pluripotent stem cells such as iPS cells and ES cells is being enthusiastically studied. However, the establishment of ES cells involves ethical issues, and iPS cells are recognized as having problems of canceration risk and length of culture period. Therefore, means using somatic stem cells such as mesenchymal stem cells and nerve stem cells having a relatively low risk of canceration, progenitor cells such as progenitor adipocytes and progenitor myocardial cells having a relatively short differentiation induction period, or chondrocytes. Possibility is also being explored in parallel.

Since a large amount of high-quality cells are required for therapeutic means using somatic stem cells or the like, a culture method capable of efficiently preparing high-quality cells is required. For example, it is known that mesenchymal stem cells can be relatively easily proliferated in a petri dish by monolayer culture (also referred to as two-dimensional (2D) culture or the like).

However, in recent reports, the 2D culture method using Chare may reduce the undifferentiated ability and proliferative ability of mesenchymal stem cells with subculture, and in addition, migration ability and anti-homing effect. There is also concern that the functions of mesenchymal stem cells, such as inflammatory effects, may decline. Therefore, in the 2D culture method, which is an existing method, it is necessary to take measures such as limiting the number of passages at the time of culturing from the viewpoint of producing high-quality cells, and this restriction is applied to cells using the 2D culture method. This is a problem when culturing in a large amount, and there is a concern that the mass-produced cells maintain uniform quality. In this regard, it has been reported that the expression of markers supporting pluripotency is improved by forming cell clusters of somatic stem cells and performing suspension culture (Patent Document 1). However, the formation of a cell mass is an indispensable technique, and there is no description about the culture in the state of a single cell (single cell) that does not form the cell mass. Since it is not technically easy to form a cell mass of uniform size and the effect of the cell mass is in the research stage, it is common to administer a single cell in the current cellular medicine. In that case, it is necessary to make the cell mass into a single cell again, which may complicate the process. In addition, the shape of mesenchymal stem cells by the 2D culture method is a spindle system adhered to the plastic surface, which is significantly different from the cell shape that originally exists in the living body. Furthermore, in the 2D culture method, since the cells are treated with an enzyme such as trypsin, it is considered that the cells that have been singled and frozen after the trypsin treatment have a reduced function of adhesion factors and the like due to the cell surface.

On the other hand, the primary cultured cells used for cell therapy include not only the above-mentioned stem cells but also finally differentiated cells such as hepatic parenchymal cells. Since these hepatic parenchymal cells do not proliferate by the 2D culture method and cause a rapid decrease in function and survival even when adhered to a plate, they are currently used immediately after thawing of frozen hepatic parenchymal cells. At that time, it is suggested that not only the viability immediately after thawing but also the cell function, especially the adhesion factor on the cell surface, is lost by the enzymatic treatment such as collagenase used when isolating the liver parenchymal cells from the liver. Has been done. In fact, many donor-derived hepatic parenchymal cells prepared by collagenase treatment have reduced adhesion to collagen-coated plates.

Polysaccharides such as deacylated gellan gum (DAG) form a three-dimensional network (atypical structure) in water by assembling via metal cations (for example, divalent metal cations such as calcium ions). When cells are cultured in a liquid medium containing this three-dimensional network, the cells in the medium are trapped in this three-dimensional network and do not sink, so that the cells are uniformly suspended in a floating state without requiring shaking or rotation operations. It is possible to culture (suspended static culture) while still dispersed in the culture medium. Further, since the above-mentioned three-dimensional network can be formed without substantially increasing the viscosity of the liquid medium, the medium composition containing the three-dimensional network is also excellent in operability in subculture and the like. It has been reported that there is (Patent Document 2).

Japanese Unexamined Patent Publication No. 2018-23401 International Publication No. 2014/017513

An object of the present invention is to provide a method for improving the quality of cells.

As a result of diligent studies to solve the above problems, the present inventors cultivate mesenchymal stem cells in a single cell state in a liquid medium composition containing deacylated gellan gum and sodium alginate without forming cell clumps. By doing so, it was found that the undifferentiated state of mesenchymal stem cells, the expression level of genes involved in migration, and the secretion amount of specific cytokines can be increased. In addition, the present inventors have found that hepatocyte adhesion can be increased by culturing hepatocytes in the liquid medium composition. The present inventors have completed the present invention by further studying based on such findings.

That is, the present invention is as follows:
[1] A liquid composition comprising a deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. The liquid composition is for promoting cell function.
[2] 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, and the concentration of the acidic polysaccharide or its salt thereof. The concentration 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 deacylated gellan gum or a salt thereof is 1 or more in terms of free form. [1] The liquid composition according to the above.
[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], further containing a metal cation.
[6] The liquid composition according to [5], wherein the metal cation is a calcium ion.
[7] The cells are mesenchymal stem cells, and the promoted cell function is at least one selected from the group consisting of undifferentiated state, migratory ability, and secretory component secretory ability, [1] to The liquid composition according to any one of [6].
[8] The liquid composition according to [7], wherein the promoted cellular function is the secretory capacity of a secretory component.
[9] Secretory factors are TSG-6 (TNF-stimulated gene 6 protein), STC-1 (Stanniocalcin-1), ANG (Angiogenin), EGF (Epidermal Growth Factor), MCP-1 (Monocyte Chemotactic Protein-1). , ENA-78 (epithelial-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor), IL-6 (Interleukin-6), IL-8 (Interleukin-8), VEGF (Vascular endothelial growth factor), VEGF -At least one selected from the group consisting of D (Vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor), and TGF-β (transforming growth factor-β) The liquid composition according to [8].
[10] The liquid composition according to any one of [1] to [6], wherein the cells are hepatocytes and the cell function promoted is cell adhesion ability.
[11] Cells in a liquid composition comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide or salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. A method of promoting cell function, including culturing.
[12] The concentration of the deacylated gellan gum or a salt thereof in the liquid composition is 0.002 to 0.1 (w / v)% in terms of the deacylated gellan gum in a free form, and the concentration of the acidic polysaccharide or a salt thereof is 0.002 to 0.1 (w / v)%. The concentration 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 deacylated gellan gum or a salt thereof is 1 or more in terms of free form. [11] The method described.
[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], further containing a metal cation.
[16] The method according to [15], wherein the metal cation is a calcium ion.
[17] The cells are mesenchymal stem cells, and the promoted cellular function is at least one selected from the group consisting of undifferentiated maintenance state, migratory ability, and secretory component secretory ability, [11] to The method described in any of [16].
[18] The method according to [17], wherein the promoted cellular function is the secretory capacity of secretory components.
[19] Secretory factors are TSG-6 (TNF-stimulated gene 6 protein), STC-1 (Stanniocalcin-1), ANG (Angiogenin), EGF (Epidermal Growth Factor), MCP-1 (Monocyte Chemotactic Protein-1). , ENA-78 (epithelial-derived neutrophil-activating peptide 78), bFGF (Basic fibroblast growth factor), IL-6 (Interleukin-6), IL-8 (Interleukin-8), VEGF (Vascular endothelial growth factor), VEGF -At least one selected from the group consisting of D (Vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor), and TGF-β (transforming growth factor-β) The method described in [18].
[20] The method according to any one of [11] to [16], wherein the cell is a hepatocyte and the promoted cell function is cell adhesion ability.

According to the present invention, it is possible to promote a specific function of a cell. For example, the function of mesenchymal stem cells (eg, undifferentiated state, migration ability, secretory component) decreased due to improvement of adhesiveness (or engraftment rate) at the time of hepatocyte transplantation and repeated passages in 2D culture. It can promote (secretory capacity) and improve its quality.

Hereinafter, the present invention will be described in detail.

1. Liquid Composition The present invention comprises a deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. , A liquid composition, wherein the liquid composition is for promoting cell function, provides a liquid composition (hereinafter, may be referred to as "the liquid composition of the present invention").

The cell in the present invention is the most basic unit constituting an animal or a plant, and has a cytoplasm and various organelles inside the cell membrane as its elements. At this time, the nucleus containing DNA may or may not be contained inside the cell. For example, the animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting the living body, stem cells, precursor cells, cancer cells separated from the living body, and immortalization ability obtained from the living body. Includes cells (cell lines) that are stably maintained in vitro, cells that have been isolated from the living body and artificially modified, and cells that have been separated from the living body and artificially exchanged nuclei. Examples of somatic cells constituting the living body are not limited to the following, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monospheres, and bones. Cells, bone marrow cells, pericutaneous cells, dendritic cells, keratinocytes, fat cells, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, cartilage cells, oval cells, nervous system cells, Glia cells, neurons, oligodendrocytes, microglia, stellate glue cells, heart cells, esophageal cells, muscle cells (eg, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanin cells, hematopoietic precursor cells, and simplex. Includes nuclear cells and the like. The somatic cells include, for example, skin, kidney, spleen, adrenal, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thoracic gland, muscle, connective tissue, bone, cartilage, blood vessel. Includes cells taken from any tissue such as tissue, blood, heart, eye, brain or nerve tissue. Stem cells are cells that have the ability to replicate themselves and differentiate into other multi-lineage cells, including, but not limited to, embryonic stem cells (ES cells). , Embryonic tumor cells, embryonic germ stem cells, artificial pluripotent stem cells (iPS cells), nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, Includes hair follicle stem cells and the like. Progenitor cells are cells in the process of differentiating from the stem cells into specific somatic cells or germ cells. Cancer cells are cells that are derived from somatic cells and have acquired infinite proliferative capacity. A cell line is a cell that has acquired infinite proliferative capacity by artificial manipulation in vitro, and examples thereof are, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116. , Huh7, HEK293 (human fetal kidney cell), 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®, Vero, etc. are included.

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 may be harvested after being induced, grown or transformed from an animal or plant by subjecting them to a particular treatment. At this time, the treatment may be in vivo or in vitro. Examples of animals include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals and the like. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrel monkeys, hamsters, voles, platypus, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, and elephants. , Common marmosets, squirrel monkeys, voles, chimpanzees 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 (eg, saponin, alkaloids, berberine, scoporin, plant sterol, etc.) (eg, medicated carrots, nichinichisou, hyos, coptis, belladonna, etc.), pigments and polysaccharides used as raw materials for cosmetics and foods. Examples include plants that produce the body (eg, anthocyanin, safflower pigment, madonna pigment, saffron pigment, flavones, etc.) (eg, blueberries, red flowers, Coptis chinensis, saffron, etc.), or plants that produce the drug substance. , Not limited to them. In a preferred embodiment, the cells can be mesenchymal stem cells or hepatocytes.

In the present specification, the mesenchymal stem cell is a somatic stem cell having self-renewal ability and pluripotency to differentiate into a plurality of mesenchymal cells. The tissue from which the mesenchymal stem cells to which the liquid composition of the present invention is applied is not particularly limited, and may be any of bone marrow, fat, umbilical cord and the like.

The liquid composition of the present invention contains deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion.

The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. Allows mesenchymal stem cells to be cultured in a floating state (preferably in a floating stationary state).

"Floating" in the present invention means that cells do not adhere to the culture vessel (non-adhesive). Further, in the present invention, when culturing cells in a liquid composition, the cells do not involve external pressure or vibration on the liquid composition, or shaking, rotation operation, etc. of the liquid composition. A state in which cells are uniformly dispersed and in a floating state is called "floating static culture", and culturing cells in this state is called "floating static culture". In addition, the period that can be suspended in "floating static" is 5 minutes or more (eg, at least 5 to 60 minutes), 1 hour or more (eg, 1 hour to 24 hours), 24 hours or more (eg, 1). Sun-21 days), 48 hours or more, 7 days or more, etc., but not limited to these periods as long as the floating state is maintained.

In a preferred embodiment, the liquid composition of the present invention is capable of floating and standing cells at at least one point in a temperature range (for example, 0 to 37 ° C.) at which cells can be cultured in a non-frozen state. The liquid composition of the present invention preferably has at least one point in the temperature range of 1 to 30 ° C., more preferably at least one point in the temperature range of 15 to 30 ° C., and more preferably at least one point in the temperature range of 22 to 28 ° C. At least one point, more preferably at least one point in the temperature range of 24 to 26 ° C., most preferably at least 25 ° C., allows suspension of cells.

Whether or not floating still is possible is determined, for example, ^{by uniformly dispersing the cells to be cultured at a concentration of 2 × 10 4} cells / ml in the liquid composition to be evaluated and 10 ml in a 15 ml conical tube. Inject the cells and allow them to stand at the desired temperature (eg, 25 ° C, 37 ° C) for at least 5 minutes (eg, 1 hour or longer, 24 hours or longer, 48 hours or longer, 7 days or longer), and the cells are suspended. Can be evaluated by observing whether or not is maintained. If more than 70% of all cells are suspended, it can be concluded that the suspended state was maintained. Instead of cells, polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) may be used for evaluation.

The liquid composition of the present invention contains deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. Allows the cells to be cultured while maintaining good viability. In a preferred embodiment, the liquid composition of the present invention is a deacylated gellan gum or a salt thereof, and an acidic polysaccharide or an acidic polysaccharide thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. By containing a salt, it has a property (effect of maintaining the floating state of cells and tissues) that enables culture in a floating state of cells (preferably floating and standing).

Deacylated gellan gum is a linear polymer composed of four sugars: 1-3-linked glucose, 1-4-linked glucuronic acid, 1-4-linked glucose, and 1-4-linked rhamnose. It is a polysaccharide and is a polysaccharide represented by the following general formula (I) (where R ₁ and R ₂ are both hydrogen atoms and n is an integer of 2 or more). However, R ₁ may contain a glyceryl group and R ₂ may contain an acetyl group, but the content of the acetyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

Deacylated gellan gum is obtained by culturing gellan gum-producing microorganisms in a fermentation medium and subjecting the mucosal substances produced outside the cells to alkaline treatment to remove glyceryl groups and acetyl groups bound to 1-3 bound glucose residues. It can be produced by recovering it after acylation, drying it, crushing it, and then making it into a powder. Examples of the purification method include liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Cefadex LH-20, adsorption chromatography with activated charcoal, silica gel, etc., or thin layer chromatography. Purification can be performed by removing impurities by adsorbing and desorbing an active substance by chromatography, high-speed liquid chromatography using a reverse-phase column, or the like by combining them alone or in any order and using them repeatedly. Examples of microorganisms producing gellan gum include, but are not limited to, Sphingomonas elodea and microorganisms in which the genes of the microorganisms are modified.

Deacylated gellan gum can also be phosphorylated. The 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 a C 1-3} alkoxy group, a C _1-3 alkylsulfonyl group, a monosaccharide residue such as glucose or fructose, sucrose, or lactose. Derivatives of deacylated gellan gum substituted with oligosaccharide residues such as glycine and amino acid residues such as glycine and arginine can also be used in the present invention. Deacylated gellan gum can also be crosslinked using a crosslinker such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

Salts include alkali metal salts such as lithium, sodium and potassium; alkaline earth metal salts such as calcium, barium and magnesium; salts such as aluminum, zinc, copper and iron; ammonium salts; tetraethylammonium, tetrabutylammonium and methyl. Tertiary ammonium salts such as tributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumin, prokine, chloroprocine; glycine , Salts with amino acids such as ammonium, valine; etc.

The weight average molecular weight of the deacylated gellan gum or a salt thereof is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and further preferably 1,000,000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).

Commercially available products such as deacylated gellan gum or a salt thereof, for example, "KELCOGEL (registered trademark of CP-Kelco) CG-LA" manufactured by Sansho Co., Ltd. and "Kelcogel (CP)" manufactured by Saneigen FFI Co., Ltd.・ Kelco's registered trademark) ”etc. can be used.

A divalent metal cation (eg, calcium ion, magnesium ion, barium ion, copper ion, iron ion, zinc ion, tin ion, lead ion, etc., preferably calcium ion) that maintains a random coil state in the medium and is a divalent metal. Examples of the acidic polysaccharide or a salt thereof that can be cross-linked via ions include alginic acid (including oligoarginic acid (also referred to as “arginate oligosaccharide”)), pectin, pectinic acid, and salts thereof. It can be, preferably an arginic acid (including oligoarginic acid) or a salt thereof.

Alginic acid is a polysaccharide having a structure in which both uronic acids, α1-4 bound L-glucuronic acid and β1-4 bound D-mannuronic acid, are linearly polymerized.

Alginic acid or a salt thereof can be extracted and purified from brown algae represented by kelp and wakame seaweed by performing an ion exchange reaction with the carboxyl group of alginic acid. Since alginic acid in the algae forms an insoluble salt with polyvalent cations such as calcium ions, it is extracted to the outside of the algae by ion-exchange with Na to obtain water-soluble sodium alginate. Further, by adding an acid to an aqueous solution of sodium alginate, insoluble alginic acid is coagulated and precipitated, and by isolating the coagulated and precipitated alginic acid, purified alginic acid can be obtained.

Salts include alkali metal salts such as lithium, sodium and potassium; alkaline earth metal salts such as calcium, barium and magnesium; salts such as aluminum, zinc, copper and iron; ammonium salts; tetraethylammonium, tetrabutylammonium and methyl. Tertiary ammonium salts such as tributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumin, prokine, chloroprocine; glycine , Salts with amino acids such as ammonium, 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 a salt thereof is, for example, 300 to 5,000,000, preferably 300 to 1,000,000, more preferably 300 to 500,000, still more preferably 300 to 100,000, and most preferably 300 to 10,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).

As alginic acid (including oligo alginic acid) or a salt thereof, commercially available products such as the following products can also be used.
Kimika Co., Ltd .:
Kimika Argin Series IL-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
Argitex Series LL, L, M, H
Kikkoman Biochemifa Co., Ltd .:
Duck Argin NSPH2R, NSPHR, NSPMR, NSPLR, NSPLLR
Sansho Co., Ltd .:
Skogin, San Argin Hokkaido Mitsui Chemicals, Inc .:
Alginate oligosaccharide ALGIN

Deacylated gellan gum and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions are tautomers produced by intra-ring or extra-ring isomerization. It may be present in the form of geometric isomers, tautomers or mixtures of geometric isomers, or mixtures thereof. Acidic polysaccharides that maintain a random coil state in deacylated gellan gum and divalent metal cation media and can be crosslinked via divalent metal ions have asymmetric centers, whether or not they result from isomerization. In some cases, they may be present in the form of split optical isomers or mixtures containing them in any proportion.

Deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion, are metal cations in the liquid composition (eg, a salt thereof). , Divalent metal cations such as calcium ions) to form a three-dimensional network (atypical structure). It is known that polysaccharides form microgels via metal cations (for example, JP-A-2004-129596), and the amorphous structure also includes the microgel as one embodiment. 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 crosslinked via a divalent metal ion are aggregated via a metal cation. As one aspect thereof, a film-like structure can be mentioned. In the liquid composition of the present invention, the deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion are used. It contains a three-dimensional network (atypical structure) formed by aggregating through metal cations (eg, divalent metal cations such as calcium ions). When cells are suspended and cultured in the liquid composition of the present invention, the cells suspended in the liquid composition are trapped in this three-dimensional network and do not settle, so that shaking, rotation operation, etc. are not required. , The cells can be cultured (suspended static culture) while being uniformly dispersed in a suspended state. The liquid composition of the present invention preferably contains the three-dimensional network (atypical structure) in a uniformly dispersed manner.

In a preferred embodiment, the formation of the three-dimensional network (atypical structure) is not particularly limited as long as the liquid composition of the present invention is a plastic fluid and has fluidity. In particular, it means that the viscosity of the liquid composition does not exceed 10000 mPa · s. At this time, the viscosity of the liquid composition is 5000 mPa · s or less, preferably 1000 mPa · s or less, and more preferably 100 mPa · s or less at 25 ° C.

The viscosity of the liquid composition can be measured, for example, by the method described in Examples described later. Specifically, E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1 ° 34'x R24, rotation speed 100 rpm) under 25 ° C conditions. Can be measured using.

The liquid composition of the present invention is other than "deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion". May contain a polysaccharide thereof or a salt thereof. The polysaccharide is preferably an acidic polysaccharide having an anionic functional group. The acidic polysaccharide is not particularly limited as long as it has an anionic functional group in its structure, but is, for example, a polysaccharide having uronic acid (for example, glucuronic acid, isulonic acid, galacturonic acid, mannuronic acid) in its structure. Polysaccharides having a sulfate group or a uronic acid group as part of the above, or polysaccharides having both structures, not only naturally obtained polysaccharides, but also polysaccharides produced by microorganisms, genetically engineered. It also includes the produced polysaccharides or the polysaccharides artificially synthesized using enzymes. More specifically, hyaluronic acid, native gellan gum, lambzan gum, daiyutan gum, xanthan gum, carrageenan, zantan gum, hexuronic acid, fucoidan, pectin, pectinic acid, pectinic acid, heparan sulfate, heparan, heparitin sulfate, keratosulfate, chondroitin sulfate. , Dermatan sulfate, ramnan sulfate, or salts thereof.

The concentration of the deacylated gellan gum or a salt thereof in the liquid composition of the present invention (equivalent to deacylated gellan gum in a free form) 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)%, and even more preferably 0.0033 to 0.0066 (w / v)%.

Concentration of an acidic polysaccharide (eg, arginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium in the liquid composition of the present invention and can be crosslinked via a divalent metal ion (free form equivalent). Is, for example, 0.004 to 0.2 (w / v)%, preferably 0.004 to 0.02 (w / v)%, more preferably 0.004 to 0.015 (w / v)%, and even more preferably 0.005 to 0.015 (w /). v)%, more preferably 0.0066 to 0.0133 (w / v)%.

The concentration of deacylated gellan gum or a salt thereof is preferably 0.002 (w / v)% or more, preferably 0.003 (w / v)% or more, from the viewpoint of ensuring a sufficient action of suspending cells. On the other hand, if this concentration is too high, the floating action may become stronger and the cell recovery rate may decrease, or the handleability of the medium itself may decrease. Therefore, 0.1 (w / v)% or less, preferably 0.009 ( It is preferably w / v)% or less. The concentration of an acidic polysaccharide (eg, alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion maintains the floating state of the cell by shearing force. From the viewpoint of ensuring the property of rapidly eliminating the effect (vulnerability to the shear force of the effect of maintaining the floating state of cells), 0.004 (w / v)% or more, preferably 0.005 (w / v)% or more. It is preferable to do so. On the other hand, if this concentration is too high, gelation may occur, so the concentration should be 0.2 (w / v)% or less, preferably 0.02 (w / v)% or less, and more preferably 0.015 (w / v)% or less. Is preferable.

Deacylated gellan gum or a salt thereof contained in the liquid composition of the present invention and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion (eg,). The mass ratio of alginic acid) or its salt (in terms of free form) is based on deacylated gellan gum or 1 part by mass of its salt from the viewpoint of achieving the property of rapidly eliminating the effect of maintaining the floating state of cells by shearing force. On the other hand, 1 part by mass or more, preferably 2 parts by mass or more of an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. And. In one embodiment, an acidic polysaccharide (eg, alginic acid) that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion with respect to 1 part by mass of deacylated gellan gum or a salt thereof. Alternatively, the salt thereof may be, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, and more preferably 1 to 2 parts by mass.

The compound concentration in the liquid composition can be calculated by the following formula.

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

The liquid composition of the present invention is an acidic polysaccharide that can maintain a random coil state in a deacylated gellan gum or a salt thereof, and a divalent metal cation medium at the above content, and can be crosslinked via a divalent metal ion. For example, the inclusion of alginic acid) or a salt thereof has the effect of maintaining good viability of cultured cells. The liquid composition of the present invention is an acidic polysaccharide that can maintain a random coil state in a deacylated gellan gum or a salt thereof, and a divalent metal cation medium at the above content, and can be crosslinked via a divalent metal ion. For example, by containing alginic acid) or a salt thereof, it has the effect of maintaining the floating state of cells.

The liquid composition of the present invention maintains a random coil state in a deacylated gellan gum or a salt thereof, and a divalent metal cation medium at the above content, and is an acidic polysaccharide that can be crosslinked via a divalent metal ion (eg,). , Alginic acid) or a salt thereof, the effect of maintaining the floating state of cells is rapidly lost by the shearing force such as pipetting and filter filtration (against the shearing force of the effect of maintaining the floating state of cells). Vulnerability) is also provided.

The liquid composition of the present invention is a deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. The salt contains a three-dimensional network (atypical structure) formed by aggregating through metal cations (eg, divalent metal cations such as calcium ions), which has the effect of maintaining the floating state of cells. However, by containing an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion, the three-dimensional network can be formed. It becomes vulnerable to a chelating agent or a shearing force, and the shearing force such as pipetting or filter filtration easily destroys this three-dimensional network, and the effect of maintaining the floating state of cells is rapidly lost. Deacylated gellan gum has a relatively linear structural unit, and multiple deacylated gellan gum chains are bundled in a liquid composition to form a tight and stable three-dimensional network. While this three-dimensional network is not easily destroyed by a chelating agent, pipetting, filter filtration, etc., it contains both α1-4 bound L-glucuronic acid and β1-4 bound D-mannuronic acid. Add an acidic polysaccharide (eg, arginic acid) or a salt thereof, which can maintain a random coil state in a divalent metal cation medium having a relatively bulky structure and can be crosslinked via a divalent metal ion, to the liquid composition. Then, it is considered that the three-dimensional network becomes vulnerable to shearing forces such as pipetting and filter filtration by suppressing the bundling of deacylated gellan gum, but this theory is not particularly bound.

As described above, in the liquid composition of the present invention, deacylated gellan gum or a salt thereof, and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. (Eg, alginic acid) or a salt thereof aggregates via a metal cation in a liquid composition (eg, a divalent metal cation such as calcium ion) to form a three-dimensional network (atypical structure). , The liquid composition of the present invention contains metal cations such as divalent metal cations (calcium ion, magnesium ion, zinc ion, iron ion, copper ion and the like), preferably calcium ion. The metal cation can be used in combination of two or more, for example, calcium ion and magnesium ion, calcium ion and zinc ion, calcium ion and iron ion, calcium ion and copper ion. Those skilled in the art can appropriately determine the combination. The metal cation concentration in the liquid composition of the present invention is 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM, but is not limited thereto.

Destruction of the three-dimensional network (loss of the property of maintaining the floating state of cells and tissues) due to shearing force such as pipetting and filter filtration is a reversible reaction. Fragments of a three-dimensional network (atypical structure) destroyed by shear forces reassemble via metal cations (eg, divalent metal cations such as calcium ions), resulting in a three-dimensional network (non-atypical). This is because the standard structure) is regenerated.

The liquid composition of the present invention contains a medium used for cell culture. The liquid composition of the present invention is acidic that can maintain a random coil state in a medium used for cell culture, deacylated gellan gum or a salt thereof, and a divalent metal cation medium, and can be cross-linked via divalent metal ions. It can be prepared by mixing with a polysaccharide (eg, alginic acid) or a salt thereof.

In the liquid composition of the present invention, components that can be added to the medium in cell culture can be further added. For example, fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenate, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins. , Various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices and various cell adhesion molecules. Examples of the cytokine added to the liquid composition of the present invention include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), and 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 stimulator (G-CSF), monocytic colony stimulator (M-CSF), granulocyte-macrophage colony stimulator (GM-CSF), stem cell factor ( SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitor (LIF), oncostatin M (OM), erythropoetin (EPO), thrombopoetin (TPO), etc., but are not limited to these.

The hormones added to the liquid composition of the present invention include melatonin, serotonin, tyrosin, triiodotyronin, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenal cortex stimulating hormone, angiotensinogen and angiotensin. Antidiuretic hormone, atrial sodium diuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicular stimulating hormone, gastrin, grelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placenta Sex lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanin cell stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, tyrotropin-releasing hormone, Cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstendione, dihydrotestosterone, estradiol, estron, estriol, progesterone, calcitriol, calcidiol, prostaglandin, leukotriene, prostacycline, thromboxane, prolactin-releasing hormone, lipotropin , Brain sodium diuretic peptide, hormonal peptide Y, histamine, endoserin, pancreatic polypeptide, renin, and enchefalin, but are not limited to these.

Growth factors added to the liquid composition of the present invention include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), Epithelial cell growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8) , 9), Nerve Cell Growth Factor (NGF), Hepatocyte Growth Factor (HGF), Leukemia Inhibitor (LIF), Prosthesis Nexin I, Prosthesis Nexin II, Thrombotic Growth Factor (PDGF), Cholinergic Differentiation Factor (CDF), chemokine, Notch ligand (such as Delta1), Wnt protein, angiopoetin-like protein 2, 3, 5 or 7 (Angpt 2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor-binding protein (IGFBP), Pleiotrophin, etc., but are not limited to these.

It is also possible to add artificially modified amino acid sequences of these cytokines and growth factors by gene recombination technology. Examples thereof include IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and a soluble IL-6 receptor).

Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, etc. Various elastin, various proteoglycans, various cadoherin, desmocholine, desmograin, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, Examples thereof include chitosan, cepharose, hyaluronic acid, alginate gel, various hydrogels, and cut fragments thereof.

Examples of antibiotics added to the liquid composition of the present invention include sulfa preparations, penicillin, pheneticillin, methicillin, oxacillin, chloroxacillin, dicloxacillin, flucloxacillin, naphthicillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin. , Piperacillin, azurocillin, mexulocillin, mesylinum, andinocillin, cephalosporins and their derivatives, oxophosphate, amoxicillin, temafloxacin, nalidixic acid, pyromidic acid, cyprofloxane, synoxacin, norfloxacin, perfloxacin, rosaxacin, offloxin Sulbactam, clavulinic acid, β-bromopenicillic acid, β-chloropenicillic acid, 6-acetylmethylene-penicillin acid, cephalosporin, sultamipicillin, adinocillin and sulbactam formaldehyde / food rate esters, tazobactam, aztreonum, sulfazetin, isosul Examples include phasetin, nocardicin, m-carboxyphenol, methyl phenylacetamide phosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxicillin, metacycline, and minocycline.

In a preferred embodiment, the liquid composition of the present invention contains a metal cation (for example, a divalent metal cation (calcion ion, magnesium ion, zinc ion, iron ion, copper ion, etc.), preferably calcium ion). Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion contain a metal cation. When mixed with the liquid composition, it aggregates via metal cations (eg, divalent metal cations such as calcium ions) in the liquid composition to form a three-dimensional network (atypical structure). The concentration of metal cations (preferably calcium ions) in the liquid composition is acidic that can maintain a random coil state in deacylated gellan gum or a salt thereof, and a divalent metal cation medium and can be crosslinked via divalent metal ions. The concentration is not particularly limited as long as the concentration of the polysaccharide (eg, alginic acid) or a salt thereof is sufficient to aggregate via the metal cation and form a three-dimensional network (atypical structure), but the present invention is not particularly limited. , 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. A liquid composition containing the metal cation, a deacylated gellan gum or a salt thereof, and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion (eg, alginic acid). ) Or a salt thereof, or a liquid composition free of the metal cation, a deacylated gellan gum or a salt thereof, and a divalent metal cation medium that maintains a random coil state and is divalent. An acidic polysaccharide (eg, alginic acid) that can be crosslinked via metal ions or a salt thereof may be mixed, and then a separately prepared aqueous solution containing the metal cation may be added to the mixed solution.

In addition to the above composition, the liquid composition of the present invention may contain various components having a cell life-prolonging effect when culturing cells. The components include saccharides (excluding polysaccharides) (eg, monosaccharides (glucose, etc.), disaccharides), antioxidants (eg, SOD, vitamin E, glutathione, polyphenols), hydrophilic polymers (eg, eg). Polyvinylpyrrolidone), chelating agents (eg, EDTA), sugar alcohols (eg, mannitol, sorbitol), glycerol and the like, but are not limited thereto. In one embodiment, the liquid composition of the present invention comprises saccharides (excluding polysaccharides) (eg, monosaccharides (glucose, etc.), disaccharides), antioxidants (eg, SOD, vitamin E, glutathione, polyphenols). Contains at least one compound selected from the group consisting of hydrophilic polymers (eg, polyvinylpyrrolidone), chelating agents (eg, EDTA), sugar alcohols (eg, mannitol, sorbitol), and glycerol.

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

In one embodiment, the liquid composition of the present invention does not have to contain a cryoprotectant. Examples of the cryoprotectant include DMSO, glycerol, ethylene glycol, trimethylene glycol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, albumin and the like. In one embodiment, the liquid composition of the invention is selected from the group consisting of DMSO, glycerol, ethylene glycol, trimethylene glycol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, and albumin at least. Does not contain one compound.

Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion are added to the above liquid medium. When added, first in a suitable solvent, deacylated gellan gum or a salt thereof, and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions ( For example, alginic acid) or a salt thereof is dissolved or dispersed (this is referred to as a medium additive). The final deacylated gellan gum or salt thereof in the liquid composition, and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions (eg, alginic acid). ) Or the salt thereof may be added to the liquid medium so that the concentration thereof becomes the concentration detailed above. A medium additive containing deacylated gellan gum or a salt thereof and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. The media additives to be included may be prepared separately and each may be added to the liquid medium, deacylated gellan gum or a salt thereof, and maintain a random coil state in a divalent metal cation medium and divalent. Maintain a random coil state in medium additives (ie, deacylated gellan gum or its salts, and divalent metal cation media) containing both acidic polysaccharides (eg, alginic acid) that can be crosslinked via metal ions or salts thereof. , And an acidic polysaccharide (eg, a mixture of alginic acid) or a salt thereof that can be crosslinked via a divalent metal ion may be prepared and added to the liquid medium. Preferably, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. A medium additive containing (ie, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion or the like. A mixture of salts) is prepared and added to the liquid medium.

Here, examples of suitable solvents used for the preparation of the medium additive include water, physiological saline, aqueous solvents such as PBS, dimethylsulfoxide (DMSO), methanol, ethanol, butanol, propanol, glycerin, propylene glycol, butylene. Examples include, but are not limited to, hydrophilic solvents such as various alcohols such as glycol. At this time, deacylated gellan gum or a salt thereof in the medium additive, and an acidic polysaccharide (eg, alginic acid) that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions or The concentration of the salt is preferably, for example, 10 to 500 times, preferably about 25 to 100 times, the final concentration in the liquid composition of the present invention described in detail above.

Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion, if necessary. It may be sterilized. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, high-pressure steam sterilization (autoclave sterilization), and filter sterilization. The material of the filter part when performing filter sterilization (hereinafter, also referred to as filtration sterilization) is not particularly limited, but for example, glass fiber, nylon, PES (polyether sulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose. Examples thereof include mixed esters, cellulose acetate, polytetrafluoroethylene and the like. 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 are deacylated gellan gum or salts thereof, and acidic polysaccharides (eg, alginic acid) or salts thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions. May be carried out in a solid state or in a solution state.

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

The combination of high-pressure steam sterilization treatment conditions is
For example, 105-135 ° C, 0.12-0.32 MPa, 1-60 minutes;
Preferably, it is 115 ° C. to 130 ° C., 0.17 to 0.27 MPa, 5 to 45 minutes;
More preferably, it is 118 to 123 ° C. (eg, 121 ± 1 ° C.), 0.19 to 0.23 MPa (eg, 0.21 ± 0.1 MPa), 15 to 25 minutes (eg, 20 ± 1 minute).

A solution or dispersion of deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. An acidic polysaccharide that can be added to a liquid medium to maintain a random coil state in a deacylated gellan gum or a salt thereof, and a divalent metal cation medium, and to be crosslinked via a divalent metal ion (a deacylated gellan gum or a salt thereof). For example, arginic acid) or a salt thereof is aggregated via a metal cation (for example, a divalent metal cation such as calcium ion) to form a three-dimensional network (atypical structure), and the liquid of the present invention is formed. The composition can be obtained. The medium is usually deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. Deacylated gellan gum or salts thereof, and divalent metal cation media, as they contain sufficient concentrations of metal cations (eg, calcium ions) to aggregate and form a three-dimensional network (atypical structure). The liquid composition of the present invention is simply added to a liquid medium with a solution or dispersion of an acidic polysaccharide (eg, arginic acid) or a salt thereof that can maintain a random coil state and can be crosslinked via divalent metal ions. Can be obtained. Alternatively, the medium additive of the present invention (deacylated gellan gum or a salt thereof, and an acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion (eg, alginic acid). Alternatively, the medium may be added to a solution or dispersion of a salt thereof. In addition, the liquid compositions of the invention are deacylated gellan gums or salts thereof, and acidic polysaccharides that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions (eg, arginic acid). ) Or a salt thereof and a medium component (powdered medium or concentrated medium) can be prepared by mixing them in an aqueous solvent (for example, water containing ion-exchanged water, ultrapure water, or the like). In the mixing mode, (1) a liquid medium and a medium additive (solution) are mixed, (2) deacylated gellan gum or a salt thereof is mixed in the liquid medium, and a random coil state is maintained in a divalent metal cation medium. And add a solid (powder, etc.) of an acidic polysaccharide (eg, alginic acid) or a salt thereof that can be crosslinked via divalent metal ions, (3) Mix the powder medium with the medium additive (solution), ( 4) Of acidic polysaccharides (eg, alginic acid) or salts thereof that can maintain a random coil state in powdered media and deacylated gellan gum or salts thereof, and can be crosslinked via divalent metal ions. Examples include, but are not limited to, mixing a solid (powder or the like) with an aqueous solvent. Distribution of deacylated gellan gum or salts thereof in liquid compositions, and acidic polysaccharides (eg, alginic acid) or salts thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions. The aspect of (1) is preferable in order to prevent non-uniformity.

Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion as a solvent (eg, water). , Aqueous solvents such as liquid media), or deacylated gellan gum or salts thereof, and acidic polysaccharides capable of maintaining a random coil state in a divalent metal cation medium and cross-linking via divalent metal ions. When dissolving (eg, alginic acid) or a salt thereof, and a powder medium in a solvent, the mixed solution may be heated to promote the dissolution. Examples of the heating temperature include 80 ° C. to 130 ° C., preferably 100 ° C. to 125 ° C. (eg, 121 ° C.) for heat sterilization. After heating, an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in the obtained deacylated gellan gum or a salt thereof and a divalent metal cation medium and can be crosslinked via a divalent metal ion. Cool the solution to room temperature. By adding the above-mentioned metal cations (eg, divalent metal cations such as calcium ions) to the solution (eg, by adding the solution to a liquid medium), deacylated gellan gum or a salt thereof, and two. Acidic polysaccharides (eg, arginic acid) or salts thereof that can maintain a random coil state in a valent metal cation medium and can be crosslinked via divalent metal ions are metal cations (eg, divalent metal cations such as calcium ions). By assembling through the above, a three-dimensional network (atypical structure) is formed, and the liquid composition of the present invention can be obtained. Alternatively, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion are described above. When dissolved in a solvent containing a metal cation (eg, a divalent metal cation such as calcium ion) (eg, an aqueous solvent such as water, liquid medium), heating (eg, 80 ° C to 130 ° C, preferably 100 ° C to 125). Deacylated gellan gum or a salt thereof, and a random coil state in a divalent metal cation medium are also maintained by cooling the resulting solution to room temperature at ° C (eg, 121 ° C), and the divalent metal. A three-dimensional network (atypical structure) by assembling acidic polysaccharides (eg, alginic acid) or salts thereof that can be crosslinked via ions via metal cations (eg, divalent metal cations such as calcium ions). Body) is formed.

Since deacylated gellan gum or a salt thereof has a relatively linear structural unit, it is difficult to dissolve because a plurality of sugar chains are bundled when added to a solvent (eg, water). However, when an acidic polysaccharide (eg, arginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion is added thereto, α1-4 bonded L- Since it has a relatively bulky structure due to the inclusion of both uronic acid of glucuronic acid and β1-4 bound D-mannuronic acid, the bundling of deacylated gellan gum or a salt thereof is suppressed and it dissolves relatively easily. It will be like. Therefore, an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a deacylated gellan gum or a salt thereof and a divalent metal cation medium and can be crosslinked via a divalent metal ion should be heated. It can be dissolved in a solvent (eg, an aqueous solvent such as water, liquid medium, etc.) at a relatively low temperature (eg, 0 to 37 ° C, preferably 10 to 30 ° C).

The method for producing the liquid composition of the present invention is exemplified, but the present invention is not limited thereto.

Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion in ion-exchanged water or ultrapure water. Add to pure water. Then, a temperature at which an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a deacylated gellan gum or a salt thereof and a divalent metal cation medium and can be crosslinked via a divalent metal ion can be dissolved (eg, alginic acid). For example, stir at 5 to 60 ° C., preferably 5 to 40 ° C., more preferably 10 to 30 ° C.) to dissolve until a transparent state is obtained.

After dissolution, allow to cool with stirring as necessary, and sterilize (for example, autoclave sterilization at 121 ° C for 20 minutes, filter filtration). While stirring (for example, a homomixer, etc.) any medium, the sterilized aqueous solution is added to the medium and mixed so as to be uniform with the medium. The method for mixing the aqueous solution and the medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer.

Deacylated gellan gum or a salt thereof and an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion are uniformly contained in a liquid medium. In a conical tube, for example, a liquid medium is placed in a conical tube, and the stirring state is maintained by vortexing, etc. An aqueous solution of an acidic polysaccharide (eg, alginic acid) or a salt thereof that can maintain a random coil state and can be crosslinked via divalent metal ions may be vigorously flushed into the liquid medium. By using the media preparation kit (Nissan Chemical FCeM ^TM -series Preparation Kit), the random coil state is maintained in the deacylated gellan gum or its salt, and the divalent metal cation medium, and cross-linking is performed via the divalent metal ion. A three-dimensional network (atypical structure) formed by assembling acidic polysaccharides (eg, alginic acid) or salts thereof via metal cations (eg, divalent metal cations such as calcium ions). , The liquid composition of the present invention, which is uniformly dispersed, can be easily prepared.

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

In the present specification, "cell function" means a function possessed by a cell. Cellular functions include, but are not limited to, proliferative, adhesive, differentiating, maintaining undifferentiated state, controlling differentiating state, secretory component secretory ability, and the like.

In one embodiment, the liquid composition of the present invention can be applied to mesenchymal stem cells. Although mesenchymal stem cells are known to have various functions, by culturing mesenchymal stem cells in the liquid composition of the present invention, the undifferentiated state, migration ability, and secretion of mesenchymal stem cells The amount of factor secretion can be promoted.

In the present specification, "promoting the undifferentiated state of mesenchymal stem cells" means that the mesenchymal stem cells shift to a more preferable state as an undifferentiated state and the differentiation of the cells is suppressed. means. More specifically, it means that, for example, the transcription amount of an undifferentiated marker such as OCT4 or NANOG gene in mesenchymal stem cells is higher than the transcription amount before application of the liquid composition of the present invention. In one embodiment of the present invention, when the transcription amount of the OCT4 gene or NANOG gene is used as an index for promoting the undifferentiated state of mesenchymal stem cells, the rate of increase in the transcription amount of the OCT4 gene or NANOG gene is usually 10% or more. , Preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, It can be, but is not limited to, 260% or more, 270% or more, 280% or more, 290% or more, or 300% or more.

Further, by culturing or storing the mesenchymal stem cells in the liquid composition of the present invention, the migration ability of the mesenchymal stem cells can be promoted. The promotion of migration ability of mesenchymal stem cells can be confirmed by determining the increase of factors involved in the migration of mesenchymal stem cells by using a known method or the like. Examples of such factors include, but are not limited to, the transcription levels of the CXCR4 gene, MMP2 gene, VCAM-1 gene, Integrinα4 gene, Integrinβ1 gene, and the like.

Further, by culturing the mesenchymal stem cells in the liquid composition of the present invention, the amount of secreted factors secreted by the mesenchymal stem cells can be promoted. In the liquid composition of the present invention, "promoting the amount of secretory factor secreted by mesenchymal stem cells" means increasing the ability of mesenchymal stem cells to produce secretory factors and increasing the amount of the factor secreted extracellularly. Means that. In the present specification, the amount of secretory component increased by the application of the liquid composition of the present invention is at least 120% or more, at least 130% or more, at least, as compared with the amount of secretory component secreted as a control. 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 It means that the production / secretion amount of secretory components increases by 600% or more, at least 700% or more, at least 800% or more, at least 900% or more, and 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 cytometer method can be used.

Examples of such secretory factors include TSG-6 (TNF-stimulated gene 6 protein), STC-1 (Stanniocalcin-1), ANG (Angiogenin), EGF (Epidermal Growth Factor), and MCP-1 (Monocyte Chemotactic Protein-1). ), ENA-78 (epithelial-derived neurophil-activating peptide 78), bFGF (Basic fibroblast growth factor), IL-6 (Interleukin-6), IL-8 (Interleukin-8), VEGF (Vascular endothelial growth factor), VEGF-D (Vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor), TGF-β (transforming growth factor-β), HGF (Hepatocyte growth factor) (Prostaglandin E2), etc., but are not limited to these.

In another embodiment, 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 aspect, the liquid composition of the present invention can be suitably used for the preparation of hepatocytes for transplantation.

2. Methods for Promoting Cell Function The present invention also provides a method for promoting cell function (hereinafter, may be referred to as "method of the present invention"), which comprises culturing cells in the liquid composition of the present invention. ..

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

In one aspect of the method of the present invention, cell culture can be performed by suspension culture (preferably suspension static culture).

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

Hereinafter, the present invention will be described in more detail by specifically describing examples of the liquid composition of the present invention, but the present invention is not limited thereto.

[Test Example 1] Preparation of a polysaccharide mixed aqueous solution 0.67 parts by mass of sodium alginate (ALG) (Kimika Argin IL-2, manufactured by Kimika Co., Ltd.) and 0.33 parts by mass of deacylated gellan gum (DAG) (KELCOGEL CG-LA, tricrystal) (Manufactured by Co., Ltd.) is added to a glass medium bottle containing 99 parts by mass of purified water, dissolved by stirring, and then passed through a 0.22 μm sterile filter (Corning filter system 430767, manufactured by Corning) for a total of 1 A polysaccharide mixed aqueous solution having a mass% concentration was prepared.

[Test Example 2] Preparation of ALG / DAG-blended medium composition A medium composition was prepared using a medium preparation kit (Nissan Chemical FCeM (registered trademark) -series Preparation Kit). Human prepared by adding MSC Nutristem (registered trademark) XF Supplement Mix (05-200-1U, manufactured by Biological Industries) to MSC Nutristem (registered trademark) XF Basal Medium (05-200-1A, manufactured by Biological Industries). 49.5 mL of Xenofree medium for mesenchymal stem cells was dispensed into the 50 mL conical tube attached to the above kit, and the adapter cap, which is a component of the kit, was attached. The tip of the disposable syringe filled with 0.5 mL of the polysaccharide mixture obtained in Test Example 1 is fitted into the cylindrical part of the adapter cap to connect, and the plunger of the syringe is manually pressed to vigorously press the polysaccharide mixture in the syringe. Was injected into a container and instantly mixed with the medium to prepare a liquid medium composition having a final polysaccharide concentration of 0.01% (w / v) (hereinafter referred to as ALG / DAG mixed medium composition).

[Test Example 3] Gene expression analysis of human umbilical cord-derived mesenchymal stem cells statically cultured in a 3D floating state in an ALG / DAG combination medium composition Human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Co., Ltd.) ) Was seeded in the ALG / DAG mixed medium composition prepared in Test Example 2 to 100,000 cells / mL, added to a 1.5 mL tube, and left to stand at room temperature for 6 days. 350 μL (RNeasy mini kit (QIAGEN, # 74106)) of RLT solution was added to human umbilical cord-derived mesenchymal stem cells used at the time of seeding to obtain an RNA extraction solution in advance. After 6 days, transfer from 1.5 mL tube to 15 mL tube and add 20% (v / v) of chelating agent (mixed aqueous solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)). After addition, the mixture was centrifuged at 300 × g for 5 minutes, and then the culture supernatant was removed. Subsequently, 350 μL (RNeasy mini kit (manufactured by QIAGEN, # 74106)) of the RLT solution was added to prepare an RNA extraction solution. For comparison, RNA extraction solutions were obtained from human umbilical cord-derived mesenchymal stem cells that were two-dimensionally cultured for 3 days using a 10 cm culture dish in the presence of _{5% CO 2 at 37 ° C.} After adding 350 μL of 70% ethanol to the RNA extraction solution, the mixture was added to an RNeasy spin column and centrifuged at 8000 × g for 15 seconds. Subsequently, 700 μL of RW1 solution was added to the RNeasy spin column, and the mixture was centrifuged at 8000 × g for 15 seconds. Subsequently, 500 μL of RPE solution was added and centrifuged at 8000 × g for 15 seconds. Further, 500 μL of RPE solution was added, and the mixture was centrifuged at 8000 × g for 2 minutes. An RNase-free solution was added to the RNA present in the RNeasy spin column and eluted. Next, cDNA was synthesized from the obtained RNA using the PrimeScript RT reagent Kit (Perfect Real Time) (# RR037A, manufactured by Takara Bio Inc.). Real-time PCR was performed using the synthesized cDNA, Premix EX Taq (Perfect Real Time) (manufactured by Takara Bio Inc., # RR039A), and Taq man Probe (manufactured by Applied Bio Systems). As 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 instrument used was 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, OCT4 showing undifferentiated characteristics of human umbilical cord-derived mesenchymal stem cells that were statically suspended and stored in the ALG / DAG combination medium composition was more undifferentiated than human umbilical cord-derived mesenchymal stem cells that were cultured at midnight or two-dimensionally. , The relative increase in the expression level of the NANOG gene and the CXCR4 gene involved in homing was observed. Table 1 shows the relative values of each gene expression.

[Test Example 4] TSG-6 production of human umbilical cord-derived mesenchymal stem cells statically stored in a 3D floating state in the ALG / DAG combination medium composition Human umbilical cord-derived mesenchymal stem cells (C-12971, Takara Bio) Was inoculated to the ALG / DAG mixed medium composition prepared in Test Example 2 to 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 1.5 mL tube to 15 mL tube and add 20% (v / v) of chelating agent (mixed aqueous solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)). After addition, the mixture was centrifuged at 300 × g for 5 minutes, and then the culture supernatant was removed. After removing the culture supernatant, it contains Xenofree medium for human mesenchymal stem cells (Sample 1) or TNF-α (# 210-TA, manufactured by R & D Systems) with a final concentration of 20 ng / mL so that it becomes 60000 sells / mL. Xenofree medium for human mesenchymal stem cells (Sample 2) was added to a 15 mL tube and added to the wells of a 24-well flat-bottomed ultra-low adhesion surface microplate (# 3473, manufactured by Corning). For comparison, human umbilical cord-derived mesenchymal stem cells two-dimensionally cultured in the presence of 5% CO _{2 at} 37 ° C using a 10 cm culture dish were used as a xenofree medium for human mesenchymal stem cells at 60000 sells / mL. Seed in sample 3) or Xenofree medium for human mesenchymal stem cells (Sample 4) containing TNF-α (# 210-TA, manufactured by R & D Systems) with a final concentration of 20 ng / mL, and 24-well flat-bottomed ultra-low adhesion surface micro Added to the plate. The plates were _{cultured in a CO 2} incubator (37 ° C, 5% CO ₂ ) in a static state and continued for 1 day. On day 1, cells and medium were transferred from each well to a 15 mL tube, centrifuged at 300 xg for 3 minutes, and then the culture supernatant was collected. 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 collection of the culture supernatant and suspended. After allowing to stand at room temperature for a minute, the luminescence intensity (RLU value) was measured with Enspire (manufactured by Perkin Elmer), and the number of living cells was measured by subtracting the luminescence value of the medium alone. Subsequently, TSG-6 contained in the collected culture supernatant was quantified using ELISA. TSG-6 antibody (# sc-65886, # sc-65886) diluted to 10 μg / mL with 0.2 M carbonate-bicarbonate buffer (pH 9.2) on Maxisorp flat bottom (# 44-2404-21, manufactured by Thermo Fisher Scientific). (Santacruz) was added at 50 μL / well and allowed to stand at 4 ° C. for 24 hours. After 24 hours, add Tween-20 (# P7949, manufactured by Sigma-Aldrich) to D-PBS (-) (# 043-29791, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.05% (v / v). After adding 300 μL of the solution added to (hereinafter referred to as PBST solution), the solution was removed. This operation was repeated 3 times. 100 μL of a PBST solution containing 5% of BSA (# A2153, manufactured by Sigma-Aldrich) was added, and the mixture was allowed to stand at room temperature for 30 minutes. After discarding the solution, 300 μL of PBST solution was added and removed. This operation was repeated 3 times. Subsequently, 50 μL of TSG-6 (# 2104-TS, manufactured by R & D Systems) prepared for the calibration curve and an evaluation sample were added to each well, and the mixture was allowed to stand at room temperature for 2 hours. After discarding the solution, 300 μL of PBST solution was added and removed. This operation was repeated 3 times. 50 μL of a solution of Biotinylated anti-human TSG-6 antibody (# BAF2104, manufactured by R & D Systems) diluted to 5 μg / mL with PBST was added, and the mixture was allowed to stand at room temperature for 120 minutes. After discarding the solution, 300 μL of PBST solution was added and removed. This operation was repeated 3 times. 50 μL of a solution of Streptavidin-HRP (# ab7403, manufactured by Abcam) diluted to 200 ng / mL with PBST solution was added, and the mixture was allowed to stand at room temperature for 30 minutes. After discarding the solution, 300 μL of PBST solution was added and removed. This operation was repeated 3 times. 100 μL of substrate solution (# 52-00-03, manufactured by KPL) was added, and the mixture was allowed 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 TSG-6 concentration contained in each sample was calculated by 4-parameter logistic regression of the calibration curve. In order to calculate the amount of secretion per cell number, the relative value obtained by dividing the calculated amount of TSG-6 by the luminescence intensity was calculated.

As a result, the human umbilical cord-derived mesenchymal stem cells statically suspended and cultured in the ALG / DAG combination medium composition per cell at the time of TNF-α treatment than the two-dimensionally cultured human umbilical cord-derived mesenchymal stem cells. The results showed that the amount of TSG-6 produced was high. Table 2 shows the relative values of each sample.

[Test Example 5] Promotion of E-Cadherin adhesion of primary hepatocytes derived from cynomolgus monkeys cultured in a 3D suspension in an ALG / DAG combination medium composition

Preparation of E-Cadherin Peptide Coat Plate E-Cadherin peptide (MAPTrix®-E, manufactured by Kollodis BIOSCIENCES, # 167021, # 167061) was diluted with sterile water to 0.2 mg / mL, respectively, and a 96-well cell culture plate was used. 100 μL was added to (Corning, # 3585) and stored in a refrigerator for a whole day and night. Only sterile water was added to the peptide-free wells. The peptide solution was removed before use, washed with D-PBS (-), and then used.

Culture method and control culture method of the present invention Two frozen cynomolgus monkey primary hepatocytes (manufactured by Ina Research Co., Ltd.) were mixed and suspended in HBM medium, and two cells were used. Dispense evenly into the tubes. The cells were then centrifuged at 50 xg 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, 96-well cell culture plates (Corning, # 3585) and each E-Cadherin peptide-coated plate were seeded at 100 μL / well, and culture was continued for 2 hours at _{37 ° C. and 5% CO 2.}

Cell pellets in the other tube were suspended in 5 ml of ALG / DAG mixed medium composition (culture method of the present invention) and seeded on a 6-well ultra-low adhesion plate (Corning, # 3471) at 5 mL / well. Preculture was performed for 2 hours under 5% CO _{2 at 37 ° C.} After 2 hours, transfer from the well to a 15 mL tube and add a chelating agent (a mixed solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)) to 10% (v / v). After the addition, the remaining cells in the well were further collected with D-PBS (-), and finally 5 times the amount of D-PBS (-) was added, and then centrifuged at 50 × g for 3 minutes, and then the culture supernatant was prepared. Excluded. Precultured cell pellet was suspended in 2.5 ml of HBM medium and seeded on 96-well cell culture plates (Corning, # 3585) and each E-Cadherin peptide coated plate at 100 μL / well at 37 ° C. 5% CO ₂ A further 2 hour culture was added below.

Measurement of total cell count of seeding The control culture method was used for 2 hours after seeding, whereas the culture method of the present invention was pre-cultured with the ALG / DAG mixed medium composition and then seeded for 2 hours. Add 100 μL of reagent (Cell Titer-Glo® Luminescent Cell Viability Assay, manufactured by Promega), suspend, allow to stand at room temperature for about 10 minutes, and then use FlexStation 3 (Molecular Devices) to emit light intensity (RLU value). ) Was measured, and the number of living cells was measured by subtracting the luminescence plate of the medium only.

Measurement of Adhesive Cell Numbers Remove the culture supernatant from the wells seeded on 96-well cell culture plates (untreated with E-Cadherin peptide) and each E-Cadherin peptide-coated plate, add 200 μL of HBM medium, and remove the medium. After repeating the process twice, 100 μL of HBM medium and 100 μL of ATP reagent were added to the cells remaining for adhesion and suspended, and the cells were allowed to stand at room temperature for about 10 minutes, and then the emission intensity (RLU value) was measured with FlexStation 3. The number of adherent cells was measured by subtracting the luminescence field of the medium only. The adhesion rate was obtained 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 with the ALG / DAG combination medium composition, a clear effect of improving the adhesion of cynomolgus monkey primary hepatocytes to the plate coated with the E-Cadherin peptide was observed. The results of the adhesion rate are shown in Table 3.

[Test Example 6] Promotion of adhesion of human-derived primary hepatocytes cultured in 3D suspension in ALG / DAG combination medium composition to E-Cadherin

Preparation of E-Cadherin Peptide Coat Plate E-Cadherin peptide (MAPTrix®-E, Kollodis BIOSCIENCES, # 167021) was diluted with sterile water to 0.2 mg / mL, respectively, and a 96-well cell culture plate (Corning) was used. 100 μL was added to # 3585) and stored in a refrigerator for a whole day and night. Only sterile water was added to the peptide-free wells. The peptide solution was removed before use, washed with D-PBS (-), and then used.

Culture method and control culture method of the present invention One frozen (about 5 million cells /) human primary hepatocytes (manufactured by Xeno Tech) was mixed, suspended in HBM medium, and evenly placed in two tubes. Dispense. The cells were then centrifuged at 50 xg for 3 minutes and the cell pellet in one tube was suspended in 2.5 ml of HBM medium (control culture method). The control culture method was to inoculate a 96-well cell culture plate (Corning, # 3585), an E-Cadherin peptide-coated plate and a Type I collagen-coated plate (IWAKI, # 4860-010) at 100 μL / well at 37 ° C. Culture was continued for 2 hours under 5% CO _2.

Cell pellets in the other tube were suspended in 5 ml of ALG / DAG mixed medium composition (culture method of the present invention) and seeded on a 6-well ultra-low adhesion plate (Corning, # 3471) at 5 mL / well. Preculture was performed for 2 hours under 5% CO _{2 at 37 ° C.} After 2 hours, transfer from the well to a 15 mL tube and add a chelating agent (a mixed solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)) to 10% (v / v). After the addition, the remaining cells in the well were further collected with D-PBS (-), and finally 5 times the amount of D-PBS (-) was added, and then centrifuged at 50 × g for 3 minutes, and then the culture supernatant was prepared. Excluded. Precultured cell pellets were suspended in 2.5 ml of HBM medium and 100 μL in 96-well plates (Corning, # 3585), E-Cadherin peptide coated plates and Type I collagen coated plates (IWAKI, # 4860-010). Seed in / wells and added cultures for an additional 2 hours under 37 ° C. 5% CO _2.

Measurement of total cell count of seeding The control culture method was used for 2 hours after seeding, whereas the culture method of the present invention was used for 2 hours after pre-culturing with the ALG / DAG mixed medium composition. Add 100 μL of ATP reagent (CellTiter-Glo ^TM Luminescent Cell Viability Assay, manufactured by Promega), suspend, allow to stand at room temperature for about 10 minutes, and then adjust the emission intensity (RLU value) with FlexStation 3 (Molecular Devices). The number of living cells was measured by subtracting the luminescence column of the medium only.

Measurement of Adherent Cell Numbers Remove the culture supernatant from the wells seeded on 96-well plates (untreated with E-Cadherin peptide), E-Cadherin peptide-coated plates and Type I collagen-coated plates, add 200 μL of HBM medium, and remove the medium. After repeating the rinsing step twice, 100 μL of HBM medium and 100 μL of ATP reagent were added to the cells remaining for adhesion and suspended, and the cells were allowed to stand at room temperature for about 10 minutes, and then the emission intensity (RLU value) was set on FlexStation 3. ) Was measured, and the number of adherent cells was measured by subtracting the luminescence plate of the medium only. The adhesion rate was obtained 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 with the ALG / DAG combination medium composition, a clear effect of improving adhesion of human primary hepatocytes to E-Cadherin peptide-coated plates and type collagen plates was observed. Table 4 shows the results of the adhesion to the E-Cadherin peptide, and Table 5 shows the Type I collagen coated plate.

[Test Example 7] Preparation of ALG / DAG-blended medium composition A medium composition was prepared using a medium preparation kit (Nissan Chemical FCeM (registered trademark) -series Preparation Kit). StemFit (registered trademark) For Mesenchymal Stem Cell (manufactured by Ajinomoto Co., Inc.) was dispensed at 49.2 mL into the 50 mL conical tube attached to the above kit, and the adapter cap, which is a component of the kit, was attached. The tip of the disposable syringe filled with 0.8 mL of the polysaccharide mixture obtained in Test Example 1 is fitted into the cylindrical portion of the adapter cap to connect, and the plunger of the syringe is manually pressed to vigorously press the polysaccharide mixture in the syringe. Was injected into a container and instantly mixed with the medium to prepare a liquid medium composition having a final polysaccharide concentration of 0.016% (w / v) (hereinafter referred to as ALG / DAG mixed medium composition).

[Test Example 8] Surface marker expression analysis of human umbilical cord-derived mesenchymal stem cells statically cultured in a 3D floating state in an ALG / DAG combination medium composition Human umbilical cord-derived mesenchymal stem cells (C-12971, Takara Bio Co., Ltd.) Is suspended in the ALG / DAG combination medium composition (final concentration 0.016%) prepared in Test Example 7 so as to be 100,000 cells / mL, and 2 mL is stem full (registered trademark) 15 mL centrifuge tube (MS-90150, It was sown in Sumitomo Bakelite Co., Ltd.). After sowing, the centrifuge tube was sealed and stored at room temperature for 7 days. After 7 days, add 20% (v / v) of a chelating agent (a mixed aqueous solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)) to a cell suspension of a 15 mL centrifuge tube. After addition, the mixture was centrifuged at 300 × g for 5 minutes, and then 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), APC Mouse Anti-Human CD90 (559869, manufactured by BD Biosciences), PE Mouse Anti-Human CD34 (555822, manufactured by BD Biosciences), and FITC Anti-CD11b antibody [M1 / 70] (ab24874, manufactured by Abcam) were added. Incubated for 30 minutes at room temperature and in the dark. Negative controls include BV650 Mouse IgG1, k Isotype Control (563231, BD Biosciences), BV421 Mouse IgG1, k Isotype Control (562438, BD Biosciences), APC Mouse IgG1, κ Isotype Control (555751, BD Biosciences). , PE Mouse IgG1, κ Isotype Control (555749, manufactured by BD Biosciences), FITC Rat IgG2b, kappa monoclonal [eB149 / 10H5] --Isotype control (ab136125, manufactured by Abcam) were used. The stained cells were washed twice with SM buffer and then measured with FACSLSRFortessaX-20 (manufactured by BD Biosciences).

In addition, after 7 days of culturing in the ALG / DAG mixed medium composition, the cells obtained by adding a chelating agent in the same manner as above and centrifuging were subjected to StemFit (registered trademark) For Mesenchymal Stem Cell (manufactured by Ajinomoto Co., Inc.). The cells were turbid and seeded on a 6-well plate so as to have 1 × 10 ⁵ cells / well, and then re-seeded into a flat culture and cultured at 37 ° C. in a 5% CO ₂ environment. After 3 days, the cells were detached into single cells using a Detach kit (D13101, manufactured by Takara Bio Inc.), and then FACS measurement was performed in the same manner as above.

As a result, human umbilical cord-derived mesenchymal stem cells that had been stood and suspended for 7 days in the ALG / DAG combination medium composition maintained the expression of a positive marker indicating MSC, and no increase in the expression of a negative marker was observed. From this, it became clear that the properties of MSC were maintained. In addition, when the cells after culturing by this culture method were re-seeded in a planar culture, the cells showed a normally adhered morphology, and the expression of MSC markers was maintained without any problem. Table 6 shows the percentage of positive cells for each marker.

[Test Example 9] Surface marker expression analysis of human umbilical cord-derived mesenchymal stem cells statically cultured in a 3D floating state in an ALG / DAG combination medium composition Human umbilical cord-derived mesenchymal stem cells (C-12971, Takara Bio Co., Ltd.) Is suspended in the ALG / DAG combination medium composition (final concentration 0.016%) prepared in Test Example 7 to 100,000 cells / mL, and 2 mL is stem full (registered trademark) 15 mL centrifuge tube (MS-90150, It was sown in Sumitomo Bakelite Co., Ltd.). After sowing, the centrifuge tube was sealed and stored at room temperature for 7 days. After 3, 7, 11 and 15 days, 20% of a chelating agent (a mixed aqueous solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)) was added to a cell suspension of a 15 mL centrifuge tube. After addition to (v / v), the mixture was centrifuged at 300 × g for 5 minutes, and the culture supernatant was removed. After washing the obtained cells with SM buffer (2% FBS / PBS), use BV421 Mouse Anti-Human CD271 (562562, manufactured by BD Biosciences) or BV421 Mouse IgG1, k Isotype Control (562438, manufactured by BD Biosciences). Each was added and incubated at room temperature and in the dark for 30 minutes. The stained cells were washed twice with SM buffer and then measured with FACSLSRFortessaX-20 (manufactured by BD Biosciences).

As a result, no expression of CD271 was observed in the human umbilical cord-derived mesenchymal stem cells used in this test cultured in a plane, but the human umbilical cord-derived mesenchymal stem cells that were statically suspended and stored in the ALG / DAG combination medium composition. The proportion of positive cells increased until the 7th day, and the proportion of positive cells was maintained thereafter. The percentage of positive cells for each marker is shown in Table 7.

[Test Example 10] Preparation of additives

[Test Example 12] Preparation of polysaccharide-containing medium composition A medium composition was prepared using a medium preparation kit (Nissan Chemical FCeM (registered trademark) -series Preparation Kit). StemFit (registered trademark) For Mesenchymal Stem Cell (manufactured by Ajinomoto Co., Inc.) was dispensed in 39.4 mL into the 50 mL conical tube attached to the above kit, and the adapter cap, which is a component of the kit, was attached. The tip of the disposable syringe filled with 0.6 mL of each polysaccharide mixture obtained in Test Example 11 is fitted into the cylindrical portion of the adapter cap to connect, and the plunger of the syringe is manually pressed to vigorously mix the polysaccharide in the syringe. A liquid medium composition having a final polysaccharide concentration of 0.015% (w / v) was prepared by injecting the liquid into a container and instantly mixing it with the medium.

[Test Example 13] Gene analysis of human umbilical cord-derived mesenchymal stem cells statically cultured in a 3D suspension in a polysaccharide-containing medium composition Human umbilical cord-derived mesenchymal stem cells (C-12971, manufactured by Takara Bio Co., Ltd.) , Suspended to 100,000 cells / mL in each medium composition (final additive concentration 0.015%) prepared in Test Example 12, and seeded 2 mL on an ultra-low adhesion 6-well plate (3471, manufactured by Corning). The cells were allowed to stand at room temperature. After 4 or 7 days, transfer the cell suspension to a 15 mL centrifuge tube and add 20% (v) of a chelating agent (a mixed solution of EDTA-2Na 0.033% (w / v) and sodium citrate 0.007% (w / v)). After addition to / v), the mixture was centrifuged at 300 × g for 5 minutes, and then the culture supernatant was removed. Subsequently, 350 μL (RNeasy mini kit (manufactured by QIAGEN, # 74106)) of the RLT solution was added to prepare an RNA extraction solution. For comparison, human umbilical mesenchymal stem cells were suspended in StemFit® For Mesenchymal Stem Cell medium and coated with iMatrix-511 silk (892 021, manufactured by Matrixome) to ^{0.5 μg / cm 2.} The cells were seeded on a 6-well plate at 50,000 cells / well and _{cultured in a plane at 37 ° C. in a 5% CO 2} environment, and the cells were subcultured using a Detach kit (D13101, manufactured by Takara Bio) on the 4th day of the culture. After adding 350 μL of 70% ethanol to the RNA extract solution obtained from these cells on the 4th or 7th day of culture, the cells were added to the RNeasy spin column and centrifuged at 8000 × g for 15 seconds. Subsequently, 700 μL of RW1 solution was added to the RNeasy spin column, and the mixture was centrifuged at 8000 × g for 15 seconds. Subsequently, 500 μL of RPE solution was added and centrifuged at 8000 × g for 15 seconds. Further, 500 μL of RPE solution was added, and the mixture was centrifuged at 8000 × g for 2 minutes. An RNase-free solution was added to the RNA present in the RNeasy spin column and eluted. Next, cDNA was synthesized from the obtained RNA using the PrimeScript RT reagent Kit (Perfect Real Time) (# RR037A, manufactured by Takara Bio Inc.). Real-time PCR was performed using the synthesized cDNA, Premix EX Taq (Perfect Real Time) (manufactured by Takara Bio Inc., # RR039A), and Taq man Probe (manufactured by Applied Bio Systems). As Taq man Probe (manufactured by Applied Bio Systems), Hs04260367_gH was used for OCT4, Hs04399610_g1 for NANOG, and Hs99999905_m1 for GAPDH. The instrument used was 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, an increase in the relative expression levels of the undifferentiated OCT4 and NANOG genes was observed in all of the additives evaluated this time. Table 9 shows the relative values of OCT4 gene expression, and Table 10 shows the relative values of NANOG gene expression.

According to the present invention, for example, the adhesive ability of hepatocytes can be improved, and the function of mesenchymal stem cells, which has been deteriorated due to repeated passages in 2D culture, can be improved. In other words, the present invention can achieve improvement in engraftment rate at the time of hepatocyte transplantation and quality adjustment or quality improvement in 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), the contents of which are incorporated herein by reference in its entirety.

Claims

A liquid composition comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide or salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion.
A liquid composition in which the liquid composition is for promoting cell function.
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, and the concentration of the acidic polysaccharide or its salt is Claim 1 that the mass ratio of the acidic polysaccharide or its salt to the deacylated gellan gum or its salt is 1 or more in terms of free form, which is 0.004 to 0.2 (w / v)% in terms of free form. The liquid composition described.
The liquid composition according to 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 according to claim 3, wherein the acidic polysaccharide is alginic acid.
The liquid composition according to any one of claims 1 to 4, further containing a metal cation.
The liquid composition according to claim 5, wherein the metal cation is a calcium ion.
13. The liquid composition according to item 1.
The liquid composition according to claim 7, wherein the promoted cell function is the secretory ability of a secretory component.
Secretory factors are TSG-6 (TNF-stimulated gene 6 protein), STC-1 (Stanniocalcin-1), ANG (Angiogenin), EGF (Epidermal Growth Factor), MCP-1 (Monocyte Chemotactic Protein-1), ENA- 78 (epithelial-derived neurophil-activating peptide 78), bFGF (Basic fibroblast growth factor), IL-6 (Interleukin-6), IL-8 (Interleukin-8), VEGF (Vascular endothelial growth factor), VEGF-D ( Vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor), and TGF-β (transforming growth factor-β), which is at least one selected from the group. The liquid composition according to claim 8.
The liquid composition according to any one of claims 1 to 6, wherein the cells are hepatocytes and the promoted cell function is cell adhesion ability.
The cells are cultured in a liquid composition containing a deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be cross-linked via a divalent metal ion. A method of promoting cell function, including.
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, and the concentration of the acidic polysaccharide or its salt is Claim 11 that the mass ratio of the acidic polysaccharide or its salt to the deacylated gellan gum or its salt is 1 or more in terms of free form, which is 0.004 to 0.2 (w / v)% in terms of free form. The method described.
The method according to 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 according to claim 13, wherein the acidic polysaccharide is alginic acid.
The method according to any one of claims 11 to 14, further comprising a metal cation.
The method according to claim 15, wherein the metal cation is a calcium ion.
13. Or the method described in item 1.
The method according to claim 17, wherein the promoted cell function is the secretory ability of a secretory component.
Secretory factors are TSG-6 (TNF-stimulated gene 6 protein), STC-1 (Stanniocalcin-1), ANG (Angiogenin), EGF (Epidermal Growth Factor), MCP-1 (Monocyte Chemotactic Protein-1), ENA- 78 (epithelial-derived neurophil-activating peptide 78), bFGF (Basic fibroblast growth factor), IL-6 (Interleukin-6), IL-8 (Interleukin-8), VEGF (Vascular endothelial growth factor), VEGF-D ( Vascular endothelial growth factor-D), TIMP (Tissue inhibitors of matrix metalloproteinase), PDGF (Platelet-Derived Growth Factor), and TGF-β (transforming growth factor-β), at least one selected from the group. The method of claim 18.
The method according to any one of claims 11 to 16, wherein the cell is a hepatocyte and the promoted cell function is cell adhesion ability.