Cell preservation material

Abstract

To provide a technique of preserving cell or tissue in a non-frozen state while maintaining excellent survivability.SOLUTION: A random coil state is maintained in deacylated gellan gum or salt thereof and a divalent metal cation medium, and a cell or a tissue is preserved in a non-frozen state in a liquid composition containing acidic polysaccharides or salt thereof which can be crosslinked via divalent metal ion. The acidic polysaccharides can be alginic acid. The liquid composition can further contain metal cation such as calcium ion.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a liquid composition for preserving cells or tissues in a non-frozen state, and a method for preserving cells or tissues using the liquid composition. [Background technology]

[0002] Currently, in transplantation medical research using stem cells such as iPS cells and mesenchymal stem cells, A cell bank prepares a large amount of stem cells, freezes and preserves them, and each research institute or medical institution orders the desired stem cells in a frozen state from the cell bank stock, thaws and puts them to sleep, and differentiates them into the desired cells as necessary, and then provides them for various uses. However, in a cell bank system that operates an autologous transplant system and manages and stores a large number of allogeneic cell stocks, which is expected in the future, it is difficult to stably supply a large amount of cells suitable for transplantation with the current transportation technology that assumes freezing of cells. That is, for transplantation, it is necessary to thaw the frozen stored cells, culture them, and differentiate them into the desired cells as necessary, and provide a large amount of cells in a good state. However, if the cells must be transported in a frozen state, the transplantation facility has no choice but to order the frozen cells from the cell bank that stores and manages the cells, and perform mass culture at the facility itself. Therefore, it is necessary for the transplantation facility to have mass cell culture equipment (e.g., cell processing center (CPC)). In order to achieve this, it is necessary to store large amounts of cells in a suitable state, and in facilities that do not have such facilities, it is difficult to carry out transplantation medicine. On the other hand, cell banks usually have the technology and equipment for mass-culturing cells, so if the cell bank can prepare large amounts of cells in a good state suitable for transplantation and quickly supply the obtained cells to transplantation facilities while maintaining their good state, transplantation medicine can be carried out even in facilities that do not have mass-culturing equipment for cells. To solve this problem, a technology is essential for storing and transporting large amounts of cells while maintaining their good state without freezing.

[0003] Polysaccharides such as deacylated gellan gum (DAG) aggregate through metal cations (for example, divalent metal cations such as calcium ions) to form a three-dimensional network (an amorphous structure) in water. 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. Therefore, without the need for shaking, rotation operations, etc., the cells can be cultured (cultured by floating and static culture) while being uniformly dispersed in a floating state. Also, since it is possible to form the above-mentioned three-dimensional network without substantially increasing the viscosity of the liquid medium, the medium composition containing the three-dimensional network is also excellent in operability in subculture, etc. (Patent Document 1). This medium composition that enables floating and static culture has various excellent properties such as enhancing the growth activity of various cells, and thus is expected to be applied to a wide range of technical fields such as regenerative medicine and mass production of proteins, etc. In Patent Document 2, it is disclosed that a medium composition containing nanofibers is used for the preservation and transportation of cells and tissues.

[0004]

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a technique for preserving cells or tissues in a non-frozen state while maintaining good viability.

Means for Solving the Problems

[0007] The inventors of the present invention have conducted intensive studies to solve the above problems, and as a result, have found that cells and tissues can be stored at room temperature for a long period while maintaining good viability by suspending them in a medium composition containing deacylated gellan gum and alginic acid. When spheres are stored in the medium composition, aggregation of the spheres is avoided, and the occurrence of necrosis inside the spheres is suppressed. In the medium composition, the cells could be stored at room temperature for a long period while maintaining good viability even under vibration conditions assuming the environment during transportation. Based on these findings, further studies were conducted, leading to the completion of the present invention.

[0008] That is, the present invention is as follows: [1] A liquid composition for storing cells or tissues in a non-frozen state, comprising deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions. [2] The concentration of deacylated gellan gum or a salt thereof in the liquid composition is 0.002 to 0.01 (w / v)% in terms of free deacylated gellan gum, the concentration of the acidic polysaccharide or a salt thereof is 0.004 to 0.1 (w / v)% in terms of free form, and the mass ratio of the acidic polysaccharide or a salt thereof to deacylated gellan gum or a salt thereof is 1 or more in terms of free form, the liquid composition according to [1]. [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 calcium ion. [7] The liquid composition according to any one of [1] to [6], wherein the acidic polysaccharide or a salt thereof has been subjected to high-pressure steam sterilization treatment. Preserving cells or tissues in a non-frozen state in the liquid composition according to any one of [8] to [7] A method for preserving cells or tissues, comprising [9] The method according to [8], wherein the cells or tissues are preserved while floating in the liquid composition.

[10] The method according to [8] or [9], wherein the cells or tissues are preserved at 1°C to 30°C.

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

[10] , wherein the cells or tissues are preserved in a sealed container Method.

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

[11] , wherein the cells or tissues are preserved in an environment accompanied by vibration Method.

[13] The method according to any one of [8] to

[12] , wherein cells in a spherical state are preserved. [Advantages of the Invention]

[0009] By using the liquid composition of the present invention, cells and tissues can be stored for a long period of time while maintaining good viability in a non-frozen state, for example, at room temperature or the like. When spheres are stored in the liquid composition of the present invention, aggregation between spheres is avoided, and the occurrence of necrosis inside the spheres is suppressed.

[0010] The liquid composition of the present invention is also useful for the non-frozen transportation of cells and tissues. For example, when cells are adherently cultured on a plate and transported as they are, the original function of the cells may be reduced due to the cells detaching from the plate due to vibration during transportation. However, when the liquid composition of the present invention is used, the cells and tissues can be held in a floating state, so that damage to the cells due to detachment from the plate due to vibration during transportation can be avoided, and the cells and tissues can be stored and transported while maintaining their original functions. [Brief Description of the Drawings]

[0011]

Figure 1

Mode for Carrying Out the Invention

[0012] The present invention will be described in more detail below.

[0013] Liquid composition The present invention provides a liquid composition for preserving cells or tissues in a non-frozen state. This liquid composition enables the preservation of cells and tissues in a non-frozen state while maintaining their viability.

[0014] The cells in the present invention are the most basic units that make up animals or plants, and as its elements, they have cytoplasm and various organelles inside the cell membrane. At this time, they contain DNA The nucleus to be encapsulated may or may not be contained inside the cell. For example, cells derived from animals in the present invention include germ cells such as sperm and eggs, somatic cells constituting the living body, stem cells, progenitor cells, cancer cells separated from the living body, cells (cell lines) that are separated from the living body, acquire immortality, and are stably maintained outside the body, cells artificially genetically modified separated from the living body, cells with artificially exchanged nuclei separated from the living body, and the like. Examples of somatic cells constituting the living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, heart cells, esophageal cells, muscle cells (for example, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, and mononuclear cells. The somatic cells include cells collected from any tissue such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain or nerve tissue. Stem cells are cells that have the ability to replicate themselves and the ability to differentiate into multiple other cell lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic carcinoma cells, embryonic germ stem cells, induced 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, hair follicle stem cells, and the like. Progenitor cells are cells in an intermediate stage of differentiating from the aforementioned stem cells into specific somatic cells or germ cells. Cancer cells are cells derived from somatic cells that have acquired the ability to proliferate indefinitely. A cell line is a cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside the body. Examples include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human cervical cancer cell line), HepG2 (human hepatoma cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero, etc. are included.

[0015] The plant-derived cells in the present invention include cells separated from each tissue of the plant body, and also include protoplasts obtained by artificially removing the cell wall from the cells.

[0016] The tissue in the present invention is a structural unit in which several types of cells having different properties and functions are assembled in a certain pattern. Examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nerve tissue, etc. Examples of plant tissues include meristematic tissue, epidermal tissue, assimilating tissue, mesophyll tissue, conducting tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell mass (callus), etc.

[0017] The cells or tissues to be stored in the liquid composition of the present invention can be arbitrarily selected from the cells or tissues described above. The cells or tissues can be directly collected from animals or plants. The cells or tissues may be collected after being induced, grown, or transformed from animals or plants by performing specific treatments. At this time, the treatment may be in vivo or in vitro. Examples of animals include, for example, fish, amphibians, reptiles, birds, crustaceans, hexapods, mammals, etc. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, monkeys, hamsters, gerbils, moles, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, rhesus monkeys, chimpanzees, and humans. There is no particular limitation on the plants as long as the collected cells or tissues can be liquid-cultured. For example, plants that produce crude drugs (e.g., saponins, alkaloids, berberine, scopoletin, plant sterols, etc.) (e.g., Panax ginseng, Stellaria media, Physalis alkekengi, Coptis japonica, Atropa belladonna, etc.), plants that produce pigments and polysaccharides (e.g., anthocyanins, safflower pigments, madder pigments, saffron pigments, flavones, etc.) that are raw materials for cosmetics and foods (e.g., blueberries, safflowers, Rubia tinctorum, saffron, etc.), or plants that produce pharmaceutical precursors, etc., but are not limited thereto.

[0018] In a preferred embodiment, the liquid composition of the present invention enables the preservation of living cells and tissues in a non-frozen state while maintaining a floating state.

[0019] The floating of cells or tissues in the present invention means a state where cells or tissues do not adhere to a storage or culture container (non - adhesion). Further, in the present invention, when storing cells or tissues in a liquid composition, without external pressure, vibration, or shaking or rotation operation of the liquid composition, the state where the cells or tissues are uniformly dispersed and in a floating state in the liquid composition is called "floating and static", and storing cells or tissues in this state is called "floating and static storage". Also, the period during which floating and static can be achieved includes 5 minutes or more (e.g., at least 5 - 60 minutes), 1 hour or more (e.g., 1 hour - 24 hours), 24 hours or more (e.g., 1 day - 21 days), 48 hours or more, 7 days or more, etc., but is not limited to these periods as long as the floating state is maintained.

[0020] In a preferred embodiment, the liquid composition of the present invention enables floating and static of cells or tissues at at least one point within the temperature range (e.g., 0 - 37°C) where cells and tissues can be stored in a non - frozen state. The liquid composition of the present invention preferably enables floating and static of cells or tissues at at least one point within the temperature range of 1 - 30°C, more preferably at at least one point within the temperature range of 15 - 30°C, still more preferably at at least one point within the temperature range of 22 - 28°C of at least one point, even more preferably at at least one point within the temperature range of 24 - 26°C, and most preferably at at least 25°C.

[0021] Whether floating and static is possible or not can be evaluated, for example, by uniformly dispersing the cells to be stored at a concentration of 2×10 4 cells / ml in the liquid composition to be evaluated, injecting 10 ml into a 15 ml conical tube, and allowing it to stand at a desired temperature (e.g., 25°C, 37°C) for at least 5 minutes or more (e.g., 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more), and observing whether the floating state of the cells is maintained. When 70% or more of all the cells are in a floating state, it can be concluded that the floating state is maintained. Instead of cells, polystyrene beads (Size 500 - 600 μm, manufactured by Polysciences Inc.) can be used for evaluation.

[0022] The liquid composition of the present invention 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 can be crosslinked via divalent metal ions. The liquid composition of the present invention 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 can be crosslinked by including an acidic polysaccharide or a salt thereof, it is possible to preserve cells or tissues in a non-frozen state while maintaining good viability. In a preferred embodiment, the liquid composition of the present invention 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 can be crosslinked via divalent metal ions, thereby enabling the preservation of cells or tissues in a suspended state (preferably, suspended and static preservation) (the property of maintaining the suspended state of cells and tissues).

[0023] Deacylated gellan gum is a linear high molecular polysaccharide composed of four molecules of sugar, namely 1-3 linked glucose, 1-4 linked glucuronic acid, 1-4 linked glucose, and 1-4 linked rhamnose, and is a polysaccharide represented by the following general formula (I) (wherein R1 and R2 are both hydrogen atoms, and n is an integer of 2 or more). However, R1 may contain a glyceryl group and R2 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.

Chemical formula

[0024]

Chemical formula

[0025] Deacylated gellan gum is obtained by culturing gellan gum-producing microorganisms in a fermentation medium, subjecting the mucous substances produced outside the cells to an alkali treatment, and removing the glycerin bound to the 1-3 linked glucose residues It can be produced by recovering the product after deacylating the lu group and the acetyl group, and making it into a powder after processes such as drying and pulverization. As purification methods, for example, liquid-liquid extraction, fractional precipitation, crystallization, various ion-exchange chromatographies, gel filtration chromatography using Sephadex LH-20, etc.,

[0026] adsorption chromatography or thin-layer chromatography using activated carbon, silica gel, etc. for the adsorption and desorption treatment of active substances, or high-performance liquid chromatography using a reverse-phase column, etc. can be used alone or in any order in combination, and can be used repeatedly to remove impurities and purify. Examples of the producing microorganisms of gellan gum include, but are not limited to, Sphingomonas elodea and microorganisms obtained by modifying the genes of said microorganisms.

[0027] The hydroxyl group corresponding to R1 and / or R2 of the compound represented by the general formula (I) can be replaced with a C 1-3 alkoxy group, a C 1-3 alkylsulfonyl group, a monosaccharide residue such as glucose or fructose, an oligosaccharide residue such as sucrose or lactose, an amino acid residue such as glycine or arginine, etc. Derivatives of deacylated gellan gum can also be used in the present invention. Also, deacylated gellan gum can be crosslinked using a crosslinker such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0028] Examples of salts include salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; salts of aluminum, zinc, copper, iron, etc.; ammonium salts; quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, choline, etc.; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, chlorprocaine, etc.; salts with amino acids such as glycine, alanine, valine, etc.; and the like. Salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, chlorprocaine, etc.; salts with amino acids such as glycine, alanine, valine, etc.; and the like.

[0029] The weight average molecular weight of deacylated gellan gum or its salt is preferably from 10,000 to 50,000,000, more preferably from 100,000 to 20,000,000, and still more preferably from 1,000,000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC). The weight average molecular weight of deacylated gellan gum or its salt is preferably from 10,000 to 50,000,000, more preferably from 100,000 to 20,000,000, and still more preferably from 1,000,000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).

[0030] As the deacylated gellan gum or its salt, commercially available products such as "KELCOGEL (registered trademark of CP Kelco) CG-LA" manufactured by Sankyo Co., Ltd., and "Kelcogel (registered trademark of CP Kelco)" manufactured by San-Ei Gen F.F.I., Inc. etc. can be used.

[0031] Examples of acidic polysaccharides or their salts that maintain a random coil state in a divalent metal cation (e.g., calcium ion, magnesium ion, barium ion, copper ion, iron ion, zinc ion, tin ion, lead ion, etc., preferably calcium ion) medium and can be crosslinked via divalent metal ions include alginic acid, pectin, pectic acid, their salts, etc., and preferably alginic acid or its salt.

[0032] Alginic acid is a polysaccharide having a structure in which both uronic acids, L-glucuronic acid linked by α1-4 bonds and D-mannuronic acid linked by β1-4 bonds, are linearly polymerized.

[0033] Alginic acid or its salt can be extracted and purified from brown algae typified by kombu and wakame by performing an ion exchange reaction on the carboxyl group of alginic acid. Since the alginic acid in the algal thallus forms an insoluble salt with polyvalent cations such as calcium ions, it is extracted outside the algal thallus by ion-exchanging this with Na to form water-soluble sodium alginate. Further, by adding an acid to an aqueous solution of sodium alginate, insoluble alginic acid is coagulated and precipitated, and the purified alginic acid can be obtained by isolating the coagulated and precipitated alginic acid.

[0034] Examples of the salt include salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; salts of aluminum, zinc, copper, iron, etc.; ammonium salts; quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, and choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, and chloroprocaine; salts with amino acids such as glycine, alanine, and valine; etc. In the present invention, sodium alginate is preferably used from the viewpoint of solubility in water.

[0035] The weight average molecular weight of alginic acid or its salt is preferably from 300 to 50,000,000, more preferably from 500 to 10,000,000, and still more preferably from 1,000 to 5,000,000. For example, the molecular weight can be measured by pullulan conversion using gel permeation chromatography (GPC).

[0036] As the alginic acid or its salt, commercially available products such as the following products can also be used. Kimica Corporation: Kimica Algin 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 Algitex Series LL, L, M, H Kikkoman Biochemifa Corporation: Duck Algin NSPH2R, NSPHR, NSPMR, NSPLR, NSPLLR Sankyo Corporation: Scorgin, Sun Algin Hokkaido Mitsui Chemicals, Inc.: Alginic acid oligosaccharide ALGIN

[0037] The deacylated gellan gum and the acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions may exist in the form of tautomers, geometric isomers, tautomers or a mixture of geometric isomers generated by intra-ring or extra-ring isomerization, or a mixture thereof. The deacylated gellan gum and the acidic polysaccharide that can maintain a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, regardless of whether they are generated by isomerization, may exist in the form of resolved optical isomers or a mixture containing them in any ratio when they have asymmetric centers.

[0038] The deacylated gellan gum or its salt, and the acidic polysaccharide or its salt that can maintain a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via metal cations (for example, divalent metal cations such as calcium ions) in the liquid composition to form a three-dimensional network (an amorphous structure). It is known that polysaccharides form microgels via metal cations (for example, JP-A-2004-129596), and the above-mentioned in The fixed structure includes, as one aspect, the microgel. As one aspect of the aggregated product of a deacylated gellan gum or its salt and an acidic polysaccharide or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions through metal cations, there is a film-like structure. The liquid composition of the present invention contains a three-dimensional network (an amorphous structure) formed by the aggregation of the deacylated gellan gum or its salt and an acidic polysaccharide or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions through metal cations (for example, divalent metal cations such as calcium ions). When cells or tissues are suspended and stored in the liquid composition of the present invention, the cells or tissues suspended in the liquid composition are trapped in this three-dimensional network and do not sediment. Therefore, it is possible to store (store in a floating and static state) the cells or tissues while uniformly dispersing them in a floating state without requiring shaking, rotation operations, etc. The liquid composition of the present invention preferably contains the three-dimensional network (amorphous structure) in a uniformly dispersed manner.

[0039] In a preferred embodiment, the formation of the three-dimensional network (amorphous structure) does not substantially increase the viscosity of the liquid composition of the present invention. "Does not substantially increase the viscosity of the liquid composition" means that the viscosity of the liquid composition does not exceed 8 mPa·s. The viscosity of the liquid composition at this time is 8 mPa·s or less at 25°C, preferably 4 mPa·s or less, and more preferably 2 mPa·s or less.

[0040] The viscosity of the liquid composition can be measured, for example, by the method described in the following examples. Specifically, it can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1°34’×R24, rotation speed 100 rpm) under the condition of 25°C.

[0041] The liquid composition of the present invention may contain a polysaccharide or a salt thereof other than "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 divalent metal ions". 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. For example, it includes polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides having a sulfate group or a phosphate group in a part of their structure, or polysaccharides having both structures. It includes not only polysaccharides obtained from nature but also polysaccharides produced by microorganisms, polysaccharides produced by genetic engineering, or polysaccharides artificially synthesized using enzymes. More specifically, hyaluronic acid, native gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xantan gum, hexuronic acid, fucoidan, pectin, pectinic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratan sulfate, chondroitin sulfate, dermatan sulfate, laminan sulfate, or salts thereof are exemplified.

[0042] The concentration of deacylated gellan gum or a salt thereof (in terms of free deacylated gellan gum) in the liquid composition of the present invention is, for example, 0.002 to 0.01 (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)% is.

[0043] The concentration (in terms of free form) of an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions in the liquid composition of the present invention is, for example, 0.004 to 0.1 (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)%. It is preferably 0.0066 to 0.0133 (w / v)%.

[0044] From the viewpoint of ensuring a sufficient effect of suspending cells or tissues, the concentration of deacylated gellan gum or its salt is preferably 0.002 (w / v)% or more, preferably 0.003 (w / v)% or more. On the other hand, if this concentration is too high, the suspension effect becomes strong, which may reduce the cell recovery rate or the handleability of the medium itself. Therefore, it is preferably 0.01 (w / v)% or less, preferably 0.009 (w / v)% or less. In a divalent metal cation medium, a random coil state is Maintained, and the concentration of an acidic polysaccharide (e.g., alginic acid) or its salt that can be cross-linked via divalent metal ions is preferably 0.004 (w / v)% or more, preferably 0.005 (w / v)% or more, from the viewpoint of ensuring the property that the effect of maintaining the suspended state of cells and tissues rapidly disappears due to shear force (vulnerability of the effect of maintaining the suspended state of cells and tissues to shear force). On the other hand, if this concentration is too high, there is a risk of gelation. Therefore, it is preferably 0.1 (w / v)% or less, preferably 0.02 (w / v)% or less, more preferably 0.015 (w / v)% or less.

[0045] The mass ratio (free form equivalent) of deacylated gellan gum or a salt thereof to an acidic polysaccharide (e.g., 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, which is contained in the liquid composition of the present invention, is 1 part by mass or more, preferably 2 parts by mass or more, of an acidic polysaccharide (e.g., 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, per 1 part by mass of deacylated gellan gum or a salt thereof, from the viewpoint of achieving a property of quickly losing the effect of maintaining a floating state of cells or tissues by shear force. In one embodiment, the mass ratio of an acidic polysaccharide (e.g., 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 is, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, more preferably 1 to 2 parts by mass, per 1 part by mass of deacylated gellan gum or a salt thereof.

[0046] The compound concentration in the liquid composition can be calculated by the following formula.

[0047] Concentration [(w / v)%] = mass of compound (g) / volume of liquid composition (ml) × 100

[0048] The liquid composition of the present invention contains deacylated gellan gum or a salt thereof and a divalent hydroxyl group at the above-mentioned content. The liquid composition of the present invention contains deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a metal cation medium and can be crosslinked via divalent metal ions, in the above-mentioned amounts, and contains an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions, thereby achieving the effect of maintaining good viability of cells or tissues preserved in a non-frozen state.

[0049] The liquid composition of the present invention contains deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions. By virtue of this, it has the property that the effect of maintaining the floating state of cells and tissues is rapidly lost due to shear forces such as pipetting and filter filtration (vulnerability of the effect of maintaining the floating state of cells and tissues to shear forces).

[0050] The liquid composition of the present invention contains a three-dimensional network (an amorphous structure) formed by the aggregation of deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions through a metal cation (e.g., a divalent metal cation such as calcium ion). This produces the effect of maintaining the floating state of cells and tissues. However, due to the inclusion of an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions, the three-dimensional network becomes vulnerable to chelating agents or shear forces. Due to shear forces such as pipetting and filter filtration, this three-dimensional network is easily destroyed, and the effect of maintaining the floating state of cells and tissues is rapidly lost. Deacylated gellan gum has a relatively linear structural unit, and multiple deacylated gellan gum chains bundle in the liquid composition to form a tight and stable three-dimensional network. Therefore, this three-dimensional network is difficult to be destroyed by chelating agents, pipetting, filter filtration, etc. On the other hand, by containing both uronic acids of α1-4-linked L-glucuronic acid and β1-4-linked D-mannuronic acid, it has a relatively bulky structure When an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions is added to a liquid composition, the bundling of deacylated gellan gum is suppressed, and thus the three-dimensional network is considered to be vulnerable to shear forces such as pipetting and filter filtration. However, the present invention is not particularly limited by this theory.

[0051] As described above, in the liquid composition of the present invention, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via metal cations (e.g., divalent metal cations such as calcium ions) in the liquid composition to form a three-dimensional network (an amorphous structure). Therefore, the liquid composition of the present invention contains metal cations, for example, divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), preferably calcium ions. The metal cations can be used in combination of two or more kinds, such as calcium ions and magnesium ions, calcium ions and zinc ions, calcium ions and iron ions, calcium ions and copper ions. Those skilled in the art can appropriately determine the combination. The metal cation concentration in the liquid composition of the present invention is from 0.1 mM to 300 mM, preferably from 0.5 mM to 100 mM, but is not limited thereto. The destruction of the three-dimensional network (loss of the property of maintaining the suspended state of cells and tissues) due to shear forces such as pipetting and filter filtration is a reversible reaction. This is because the fragments of the three-dimensional network (amorphous structure) destroyed by the shear force reassemble again via metal cations (e.g., divalent metal cations such as calcium ions) to regenerate the three-dimensional network (amorphous structure).

[0052]

[0053] ​The liquid composition of the present invention preferably contains a medium (preferably a liquid medium) used for culturing cells or tissues to be preserved. In this case, the liquid composition of the present invention is prepared by mixing a medium (preferably a liquid medium) used for culturing cells or tissues to be preserved, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions.

[0054] Examples of media used for culturing cells or tissues derived from animals (e.g., mammals) include Leibovitz's L-15 Medium, Dulbecco’s Modified Eagles’s Medium (DMEM), Ham’s Nutrient Mixture F12, DMEM / F12 medium, McCoy’s 5A medium, Eagle’s Minimum Essential Medium (EMEM), alpha Modified Eagle’s Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI1640 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), MCDB131 medium, William’s Medium E, IPL41 medium, Fischer’s medium, NutriStem MSC XF (manufactured by Biological Industries), NutriStem hPSC XF (manufactured by Biological Industries) StemPro34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Lonza), X-VIVO 15 (manufactured by Lonza), HPGM (manufactured by Lonza), StemSpan H3000 (manufactured by StemCell Technologies), and the like. (manufactured by Stem Cell Technologies), StemSpan SFEM (manufactured by Stem Cell Technologies), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), StemPro hESC SFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technologies), Sf-900 II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), HFDM-1 (manufactured by Nipro), Nipro EIDF (manufactured by Nipro), BMPro (manufactured by Nipro), and the like.

[0055] When the cells or tissues are of plant origin, media such as Murashige and Skoog (MS) medium, Linsmaier and Skoog (LS) medium, White medium, Gamborg's B5 medium, Nitsch medium, Heller medium, Morel medium, etc., which are commonly used in plant tissue culture, or modified media (for example, reducing the ammonium nitrogen concentration by half, etc.) obtained by modifying these medium components to appropriate concentrations, to which plant growth regulators (plant hormones) such as auxins and, if necessary, cytokinins are added at appropriate concentrations can be mentioned as media. These media can be further supplemented with, if necessary, caseinolytic enzymes, corn steep liquor, vitamins, etc. Examples of auxins include, but are not limited to, 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), etc. Auxins can be added to the medium at a concentration of, for example, about 0.1 to about 10 ppm. Examples of cytokinins include, but are not limited to, kinetin, benzyladenine (BA), zeatin, etc. Cytokinins can be added to the medium at a concentration of, for example, about 0.1 to about 10 ppm.

[0056] To the above-mentioned medium, those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. according to the purpose. When culturing animal-derived cells or tissues, those skilled in the art can also add one or more combinations of other chemical components or biological components according to the purpose. Components added to the medium for animal-derived cells and / or tissues include fetal bovine serum, human serum, equine serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, 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, various cell adhesion molecules, etc. Examples of cytokines added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon- α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin Examples include, but are not limited to, ethyne (TPO).

[0057] Hormones added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Müllerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandin, leukotriene, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin , and enkephalin, but are not limited to these.

[0058] Growth factors added to the medium include transforming growth factor-α (TGF-α). Transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), pleiotrophin, etc. are included, but not limited thereto.

[0059] In addition, those obtained by artificially modifying the amino acid sequences of these cytokines and growth factors by genetic recombination technology can also be added. Examples thereof include IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (fusion protein of IL-6 and soluble IL-6 receptor).

[0060] Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, vitronectin, laminin-1 to 12, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, des moglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, hyaluronic acid, alginate gel, various hydrogels, and further cleavage fragments thereof, etc.

[0061] Examples of antibiotics added to the medium include sulfonamides, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, mecillinam, anginosillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rosoxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulanic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultamicillin, adenocillin and the formaldehyde-hoodrate ester of sulbactam, tazobactam, aztreonam, sulfacetamide, isosulfacetamide, nocardicin, m-carboxyphenol, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0062] In a preferred embodiment, the medium (preferably a liquid medium) contains metal cations (e.g., divalent metal cations (such as calcium ions, magnesium ions, zinc ions, iron ions, and copper ions, etc.), preferably calcium ions). When mixed with the liquid medium, deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via the metal cations (e.g., divalent metal cations such as calcium ions) in the liquid medium to form a three-dimensional network (an amorphous structure). The concentration of the metal cations (preferably calcium ions) in the medium is not particularly limited as long as it is sufficient for deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions to aggregate via the metal cations and form a three-dimensional network (an amorphous structure). For example, it is 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. The liquid medium containing the metal cations may be mixed with deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, or a medium not containing the metal cations may be mixed with deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, and then an aqueous solution containing the metal cations prepared separately may be added to the mixture.

[0063] In addition to the above-described composition, the liquid composition of the present invention may contain various components having a cell life-prolonging effect during the preservation of cells and tissues in a non-frozen state. Examples of such components include saccharides (excluding polysaccharides) (e.g., monosaccharides (such as glucose), disaccharides), antioxidants (e.g., SOD, vitamin E, glutathione, polyphenols), hydrophilic polymers (e.g., polyvinylpyrrolidone), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), glycerol, etc., but are not limited thereto. In one aspect, the liquid composition of the present invention contains at least one compound selected from the group consisting of saccharides (excluding polysaccharides) (e.g., monosaccharides (such as glucose), disaccharides), antioxidants (e.g., SOD, vitamin E, glutathione, polyphenols), hydrophilic polymers (e.g., polyvinylpyrrolidone), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), and glycerol. to do.

[0064] In addition, the liquid composition of the present invention contains deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, thereby achieving the effect of maintaining good viability of cells or tissues preserved in a non-frozen state. Therefore, when preserving cells and tissues in a non-frozen state, other components having a cell life-prolonging effect may not be included. In one aspect, the liquid composition of the present invention does not contain at least one compound selected from the group consisting of saccharides (excluding polysaccharides) (e.g., monosaccharides (such as glucose), disaccharides), antioxidants (e.g., SOD, vitamin E, glutathione, polyphenols), hydrophilic polymers (e.g., polyvinylpyrrolidone), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), and glycerol. compound.

[0065] Since the liquid composition of the present invention is for preserving cells or tissues in a non-frozen state, it may not contain a cryoprotectant. Examples of cryoprotectants include DMSO, glycerol, ethylene glycol, trimethylene glycol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, albumin, and the like. In one aspect, the liquid composition of the present invention does not contain at least one compound selected from the group consisting of DMSO, glycerol, ethylene glycol, trimethylene glycol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, and albumin.

[0066] When adding deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions to the above liquid medium, first dissolve or disperse deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions in a suitable solvent (this will be used as a medium additive). Then, add the medium additive to the liquid medium such that the final concentration of deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions in the liquid composition becomes the concentration detailed above. A medium additive containing deacylated gellan gum or a salt thereof and a medium additive containing an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions may be prepared separately and each added to the liquid medium, or a medium additive containing both deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions (i.e., a mixture of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions) may be prepared and added to the liquid medium. Preferably, a medium additive containing both deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions (i.e., a mixture of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions) is prepared and added to the liquid medium.

[0067] Here, examples of suitable solvents used for preparing the medium additive include water, physiological saline, PBS, etc. Examples of hydrophilic solvents include aqueous solvents, dimethyl sulfoxide (DMSO), methanol, ethanol, butanol, propanol, glycerin, propylene glycol, various alcohols such as butylene glycol, etc., but are not limited thereto. At this time, medium addition The concentration of deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions in the medium for adding agents is, for example, 10 to 500 times the final concentration in the liquid composition of the present invention detailed above, preferably about 25 to 100 times the concentration.

[0068] Deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions may be sterilized as necessary. The sterilization method is not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization (high-pressure steam sterilization), filter sterilization, etc. The material of the filter part when performing filter sterilization (hereinafter sometimes referred to as filtration sterilization) is not particularly limited, and examples include glass fiber, nylon, PES (polyethersulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose mixed esters, cellulose acetate, polytetrafluoroethylene, etc. The pore size 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 may be performed on deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions either in a solid state or in a solution state.

[0069] The temperature in high-pressure steam sterilization treatment is usually 105 - 135°C, preferably 115°C - 130°C, more preferably 118 - 123°C (e.g., 121 ± 1°C). The pressure during the sterilization treatment is usually 0.12 - 0.32 MPa, preferably 0.17 - 0.27 MPa, more preferably 0.19 - 0.23 MPa (e.g., 0.21 ± 0.1 MPa). The sterilization treatment time is usually 1 - 60 minutes, preferably 5 - 45 minutes, more preferably 15 - 25 minutes (e.g., 20 ± 1 minute).

[0070] The combinations of high-pressure steam sterilization treatment conditions are for example, 105 - 135°C, 0.12 - 0.32 MPa, 1 - 60 minutes; preferably, 115°C - 130°C, 0.17 - 0.27 MPa, 5 - 45 minutes; more preferably, 118 - 123°C (e.g., 121 ± 1°C), 0.19 - 0.23 MPa (e.g., 0.21 ± 0.1 MPa), 15 - 25 minutes (e.g., 20 ± 1 minute).

[0071] By adding a solution or dispersion of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions to a liquid medium, in the liquid medium, deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions aggregate via metal cations (e.g., divalent metal cations such as calcium ions), thereby forming a three-dimensional network (an amorphous structure), and the liquid composition of the present invention can be obtained. Usually, the medium contains a sufficient concentration of metal cations (e.g., calcium ions) for deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions to aggregate and form a three-dimensional network (an amorphous structure). Therefore, the liquid composition of the present invention can be obtained simply by adding a solution or dispersion of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions to the liquid medium. Alternatively, the medium may be added to the medium additive of the present invention (a solution or dispersion of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions). Furthermore, the liquid composition of the present invention is an acid that maintains 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 A gelling polysaccharide (e.g., alginic acid) or its salt and a medium component (powdered medium or concentrated medium) can also be prepared by mixing them in an aqueous solvent (e.g., water including ion-exchanged water, ultrapure water, etc.). Examples of the mixing mode include (1) mixing a liquid medium and a medium additive (solution); (2) adding a solid (powder, etc.) of a deacylated gellan gum or its salt and an acidic polysaccharide (e.g., alginic acid) or its salt that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions to a liquid medium; (3) mixing a powdered medium into a medium additive (solution); (4) mixing a powdered medium, a deacylated gellan gum or its salt, and a solid (powder, etc.) of an acidic polysaccharide (e.g., alginic acid) or its salt that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions with an aqueous solvent, etc., but are not limited thereto. To prevent the distribution of the deacylated gellan gum or its salt and the acidic polysaccharide (e.g., alginic acid) or its salt that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions in the liquid composition from becoming non-uniform, the mode of (1) is preferred.

[0072] Dissolve deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, in a solvent (e.g., an aqueous solvent such as water, a liquid medium, etc.), or when dissolving deacylated gellan gum or its salt, an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, and a powder medium in a solvent, the mixture may be heated to promote dissolution. Examples of the heating temperature include, for example, 80°C to 130°C, preferably 100°C to 125°C (e.g., 121°C) such that it is heat-sterilized. After heating, cool the obtained solution of deacylated gellan gum or its salt and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions to room temperature. By adding the above-mentioned metal cation (e.g., a divalent metal cation such as calcium ion) to the solution (e.g., by adding the solution to a liquid medium), deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via the metal cation (e.g., a divalent metal cation such as calcium ion), thereby forming a three-dimensional network (an amorphous structure), and the liquid composition of the present invention can be obtained. Alternatively, when dissolving deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions in a solvent (e.g., an aqueous solvent such as water, a liquid medium, etc.) containing the above-mentioned metal cation (e.g., a divalent metal cation such as calcium ion), heat (e.g., 80°C to 130°C, preferably or 100°C to 125°C (e.g., 121°C)), and also by cooling the obtained solution to room temperature , Deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via metal cations (e.g., divalent metal cations such as calcium ions), thereby forming a three-dimensional network (an amorphous structure).

[0073] In addition, since deacylated gellan gum or its salt has a constituent unit with a relatively linear structure, when added to a solvent (e.g., water), multiple sugar chains bundle together, making it difficult to dissolve. However, when an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions is added here, by containing both uronic acids of L-glucuronic acid linked by α1-4 bonds and D-mannuronic acid linked by β1-4 bonds, it has a relatively bulky structure, so the bundling of deacylated gellan gum or its salt is suppressed and it becomes relatively easy to dissolve. Therefore, deacylated gellan gum or its salt and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions can be dissolved in a solvent (e.g., an aqueous solvent such as water or a liquid medium) at a relatively low temperature (e.g., 0 to 37°C, preferably 10 to 30°C) without heating.

[0074] The manufacturing method of the liquid composition of the present invention is illustrated, but it is not limited thereby.

[0075] Deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions are added to ion-exchanged water or ultrapure water, and the mixture is stirred at a temperature at which the deacylated gellan gum or a salt thereof and the acidic polysaccharide (e.g., alginic acid) or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions are dissolved (e.g., 5 to 60°C, preferably 5 to 40°C, more preferably 10 to 30°C) until the mixture becomes transparent.

[0076] After dissolving, cool while stirring as necessary, and sterilize (for example, autoclave at 121°C for 20 minutes). The aqueous solution after sterilization is then subjected to agitation (e.g., clave sterilization, filter filtration) of any medium. While stirring the medium (e.g., a homomixer, etc.), the sterilized aqueous solution is added to the medium and mixed uniformly with the medium. There are no particular limitations on the method for mixing the aqueous solution with the medium, and examples of the method include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, or homogenizer.

[0077] In order to uniformly disperse the deacylated gellan gum or its salt and the acidic polysaccharide (e.g., alginic acid) or its salt which maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions in the liquid medium, for example, the liquid medium may be placed in a conical tube, kept stirred with a vortex or the like, and an aqueous solution of the deacylated gellan gum or its salt and the acidic polysaccharide (e.g., alginic acid) or its salt which maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions may be vigorously flushed into the liquid medium from a syringe equipped with a syringe needle. TMBy using the [-series Preparation Kit], a deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions can aggregate via a metal cation (e.g., a divalent metal cation such as a calcium ion) to form a three-dimensional network (an amorphous structure), and the liquid composition of the present invention in which the three-dimensional network is uniformly dispersed can be easily prepared.

[0078] After mixing, the liquid composition of the present invention may be filtered through a filter. The pore size of the filter used during the filtration process is 5 μm to 100 μm, preferably 5 μm to 70 μm, more preferably 10 μm to 70 μm.

[0079] [Method for preserving cells or tissues] Furthermore, the present invention provides a method for preserving cells or tissues, which includes preserving cells or tissues in a non-frozen state in the above liquid composition of the present invention. In a preferred embodiment, in the preservation method of the present invention, cells or tissues can be preserved or transported in a suspended state (preferably, in a suspended and static state) in the liquid composition of the present invention.

[0080] Examples of the cells and tissues to be preserved in the liquid composition of the present invention include those detailed in the above section of [Liquid Composition].

[0081] The form and state of the cells and tissues to be preserved by the method of the present invention can be arbitrarily selected by those skilled in the art. Specific examples of the state of the cells to be preserved include a state dispersed in single cells, a state where the cells adhere to the surface of a carrier, a state where the cells are embedded inside a carrier, a state where a plurality of cells aggregate to form a cell mass (e.g., a sphere), and a state where two or more types of cells aggregate to form a cell mass (e.g., a sphere). Examples include, but are not limited to, the formed state and the like. Among these states, the state in which cell aggregates (e.g., spheres) are formed has cell-cell interactions and cell structures reconstructed that are close to the in vivo environment, can preserve cell functions while maintaining them in the long term, and is relatively easy to recover cells. Therefore, it can be cited as the most preferable state for preservation by the method of the present invention. The cell aggregate preferably includes mammalian stem cells (e.g., embryonic stem cells (ES cells), embryonal carcinoma cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural 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, hair follicle stem cells, cancer stem cells) or spheres containing progenitor cells. Examples of the sphere in the present invention include those in which cells form an aggregate consisting of several tens to several hundreds of cells. Spheres can be produced by known methods.

[0082] When preserving cells in the liquid composition of the present invention, the cell concentration is not particularly limited as long as it can be preserved in a non-frozen state while maintaining good viability, but is usually 0.1×10 4 ~200×10 4 cells / ml, preferably 1×10 4 ~100×10 4 cells / ml.

[0083] In the preservation method of the present invention, a desired cell or tissue is dispersed in the liquid composition of the present invention and then placed in a sealable container. Examples of the container include, but are not limited to, flasks, plastic bags, Teflon (registered trademark) bags, tubes, culture bags, etc. To avoid leakage of the contents and contamination by bacteria or the like from the outside during preservation, the container containing the dispersion of the cells or tissue in the liquid composition of the present invention is preferably sealed.

[0084] As described above, in the liquid composition of the present invention, deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions aggregate via a metal cation (e.g., a divalent metal cation such as calcium ion), thereby forming a three-dimensional network (an amorphous structure). When cells or tissues are stored in the liquid composition of the present invention, the cells or tissues suspended in the liquid composition are trapped in this three-dimensional network and do not settle. Therefore, it becomes possible to store (store in a floating and static state) the cells or tissues while keeping them uniformly dispersed in a floating state. On the other hand, this three-dimensional network contains an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions, and thus is vulnerable to shear force. When a chelating agent is added as needed to the liquid composition of the present invention and the preservation preparation containing cells or tissues, and a shear force sufficient to break this three-dimensional network is applied by pipetting or the like, the property of floating the cells or tissues based on this three-dimensional network is rapidly lost. Therefore, at the start of preservation, by breaking the three-dimensional network (an amorphous structure) formed by the aggregation of deacylated gellan gum or its salt, and an acidic polysaccharide (e.g., alginic acid) or its salt that maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions via a metal cation (e.g., a divalent metal cation such as calcium ion) by a shear force such as pipetting or stirring, it is possible to quickly and uniformly disperse and suspend the cells and tissues in the liquid composition of the present invention.Importantly, the destruction of the three-dimensional network (amorphous structure) by shear force (the loss of the effect of maintaining the floating state of cells and tissues) is a reversible reaction. When the obtained cell or tissue suspension is allowed to stand, over time, the fragments of the destroyed three-dimensional network (amorphous structure) reassemble via metal cations (e.g., divalent metal cations such as calcium ions), the three-dimensional network (amorphous structure) is regenerated, and it shows the property of maintaining the floating state of cells and tissues again, enabling the cells and tissues to be stored while maintaining the floating state.

[0085] The temperature during storage is not particularly limited as long as the survival of the cells or tissues is maintained, but usually, it is 37°C or lower. A lower temperature can avoid the decrease in the viability of the cells or tissues during storage, but usually, it is stored at a temperature above the melting point of the liquid composition of the present invention so that the cells or tissues do not freeze. Therefore, the temperature during storage is usually 0 - 37°C, preferably 1 - 30°C, more preferably 15 - 30°C (e.g., normal temperature storage at 15 - 25°C), still more preferably 22 - 28°C, and even more preferably 24 - 26°C (e.g., 25°C).

[0086] To enable the storage of cells or tissues in a floating and static state, the temperature during storage is preferably such that the liquid composition of the present invention can keep the cells or tissues to be stored in a floating and static state.

[0087] The storage period is not particularly limited within the range where the survival state of the cells or tissues to be stored can be maintained in the liquid composition of the present invention, but it is usually 1 hour or more (e.g., 12 hours or more, 24 hours (1 day) or more, 2 days or more). The upper limit of the storage period is not particularly limited as long as the survival state of the cells or tissues to be stored can be maintained in the liquid composition of the present invention, but it is usually within 28 days (e.g., within 21 days, within 14 days, within 7 days, within 3 days). The storage period is appropriately set according to the purpose of storage. During the storage or transportation period, it is preferable that the cells or tissues are maintained in a floating and static state in the liquid composition of the present invention.

[0088] In one aspect, in an environment accompanied by vibration, cells or tissues are preserved in the liquid composition of the present invention. Examples of the "environment accompanied by vibration" include the time of transporting cells or tissues. That is, the preservation method of the present invention can also be regarded as a method of transporting cells or tissues while preserving them in a non-frozen state in the liquid composition of the present invention.

[0089] When the preservation method of the present invention is used, cells and tissues can be kept in a floating state, so that damage to cells and tissues caused by detachment from the plate due to vibration during transportation or aggregation of contacted cells and tissues due to sedimentation can be avoided, and the cells and tissues can be preserved while maintaining their original functions. When spheres are preserved by the method of the present invention, the spheres can be kept in a floating state, so that vibration during transportation and aggregation of the spheres due to vibration can be avoided, and the spheres can be preserved while maintaining their form.

[0090] Recovery of stored cells or tissues The present invention also provides a method for efficiently recovering cells or tissues from a preservation preparation obtained by preserving cells or tissues in the liquid composition of the present invention. The recovery method of the present invention is characterized by applying a shearing force to the preservation preparation.

[0091] As described above, in the liquid composition of the present invention, deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions aggregate via a metal cation (e.g., a divalent metal cation such as a calcium ion), thereby forming a three-dimensional network (an amorphous structure). When cells or tissues are preserved in the liquid composition of the present invention, the cells or tissues suspended in the liquid composition are trapped in this three-dimensional network and do not sediment. Therefore, it becomes possible to preserve (preserve by floating and standing) the cells or tissues while uniformly dispersing them in a floating state. On the other hand, this three-dimensional network contains an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions, and thus is vulnerable to shear force. When a chelating agent is added as necessary to the liquid composition of the present invention and a preservation preparation containing cells or tissues, and a shear force sufficient to destroy this three-dimensional network is applied, the property of floating cells or tissues based on this three-dimensional network is rapidly lost, and the cells or tissues are likely to sediment due to gravity. In such a state, when the preservation preparation is subjected to centrifugation, the cells or tissues contained therein easily sediment, and the liquid composition of the supernatant is removed to recover the cells or tissues.

[0092] The operation of applying a shear force to the preservation preparation is not particularly limited as long as it can destroy the three-dimensional network (amorphous structure) formed by the aggregation of deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions via a metal cation (e.g., a divalent metal cation such as a calcium ion). Examples thereof include pipetting, filter filtration, stirring, ultrasonic waves, and the like.

[0093] To apply sufficient shear force to the stored preparation, it is preferable to perform pipetting using a pipette with a relatively thin tip (the inner diameter of the tip is, for example, 5 mm or less, preferably 0.1 to 3.0 mm, more preferably 0.5 to 2.0 mm).

[0094] Also, so as to be able to quickly stir the entire stored preparation, for example, 1% or more or more, preferably 10% or more, more preferably 20% or more, still more preferably 30% or more, even more preferably 50% or more of the volume of the stored preparation is preferably inhaled and exhaled at once.

[0095] To apply sufficient shear force to the stored preparation, for example, at a flow rate of 1 ml / second or more, preferably 2 to 20 ml / second, more preferably 5 to 10 ml / second, it is preferable to perform the inhalation and / or exhalation operation in this way.

[0096] The number of pipetting times is not particularly limited as long as it is sufficient to break the above three-dimensional network, but it is usually 1 time or more, preferably 3 times or more, more preferably 5 times or more continuously performed. The more the number of pipetting times, the more surely the above three-dimensional network is broken, so it is preferable. Theoretically, there is no upper limit, but if the number of pipetting times is too large, the survival rate of cells or tissues will decrease, so it is usually 50 times or less, preferably 20 times or less, more preferably 15 times or less. The number of pipetting times is usually 1 to 50 times , preferably 3 to 20 times, more preferably 5 to 15 times.

[0097] The pore size of the filter (aperture diameter) is not particularly limited as long as it can break the above three-dimensional network, but it is usually 500 μm or less, preferably 200 μm or less, more preferably 100 μm or less. The smaller the pore size, the stronger the shear force applied to the stored preparation It can be used to more reliably destroy the above three-dimensional network. However, if it is too small, it will be difficult for the liquid composition to pass through the filter. Therefore, the pore size (aperture diameter) of the filter is usually 5 μm or more, preferably 10 μm or more, more preferably 20 μm or more, and even more preferably 40 μm or more.

[0098] The pore size of the filter is preferably such that cells or tissues in the preserved preparation can pass through. Here, "a size through which cells or tissues can pass" means a size that can allow cells or tissues to pass through while maintaining their viability. For example, not only when the pore size of the filter is larger than the diameter of the cells or tissues to be preserved, but also when cell aggregates (e.g., spheres) or tissues in the preserved preparation pass through a filter with a pore size smaller than their diameter and are divided into a plurality of cells, cell aggregates (e.g., spheres) or tissues while maintaining their viability, such a mode is also included in "a size through which cells or tissues can pass". Since the size of cells depends on the type of cells, it cannot be generally defined, but for general cells with a diameter of about 7.5 to 20 μm if so, in the state of single cells, they can easily pass through a filter with a pore size of 20 μm or more, preferably 40 μm, while maintaining good viability. Therefore, from the perspective of efficiently destroying the above three-dimensional network while maintaining good viability of cells or tissues, the pore size of the filter is preferably in the range of, for example, 20 to 200 μm, preferably 40 to 100 μm.

[0099] Examples of the filter material include polyethylene, polypropylene, polyamide (nylon), polysulfone, polypropylene, acrylic, polylactic acid, cellulose mixed ester, polycarbon, polyester, glass, etc., but it is not particularly limited. Depending on the material, the performance such as polarity, chargeability, and hydrophilicity is different, but the correlation between these performances and the recovery rate is weak, and a good recovery rate can be expected regardless of which material is used. Polyamide (nylon), polyethylene, polyester, glass, etc. are preferable from the perspective of easy availability, etc.

[0100] These filters may be commercially available products. Specific examples include Partec CellTrics filters (trademark): pore sizes of 5 μm (model number 06-04-004-2323), 10 μm (model number 06-04-004-2324), 20 μm (model number 06-04-004-2325), 30 μm (model number 06-04-004-2326), 50 μm (model number 06-04-004-2327), 100 μm (model number 06-04-004-2328), and 150 μm (model number 06-04-004-2329). Cell Strainer (trademark) manufactured by Becton Dickinson: pore sizes of 40 μm (model number 352340), 70 μm (model number 352350), and 100 μm (model number 352360), and Filcon S (trademark) manufactured by AS ONE: pore sizes of 20 μm (model number 2-7211-01), 30 μm (model number 2-7211-02), 50 μm (model number 2-7211-03), 70 μm (model number 2-7211-04), 100 μm (model number 2-7211-05), and 200 μm (model number 2-7211-06), etc.

[0101] The number of times to pass through the filter may be once, but if necessary, multiple passes through the filter can improve the recovery rate of cells or tissues. The number of times to pass through the filter is usually 1 to 10 times.

[0102] When passing through the filter multiple times, the operation of passing a preserved preparation of cells or tissues through one filter and collecting the passed suspension may be performed multiple times, or a preserved preparation of cells or tissues may be passed through a multiple filter including filter membranes stacked in multiple numbers (for example, 3 to 5 sheets). Using a multiple filter is advantageous from the viewpoint of operation efficiency. When passing through the filter multiple times, multiple filters with the same pore size may be used, or multiple filters with different pore sizes may be used in combination. Preferably, multiple (for example, 3 to 5 sheets) filters with the same pore size (for example, 40 to 100 μm) are stacked and used.

[0103] ​Examples of stirring operations include vortex, inversion mixing, magnetic stirrer, paddle, etc. The speed of the vortex is, for example, 200 to 3,000 rpm.

[0104] When applying shear force to the stored preparation, a chelating agent may be added to the culture preparation as necessary. By adding a chelating agent, metal cations (preferably divalent metal cations such as calcium ions and magnesium ions) are removed from the three-dimensional network contained in the liquid composition, and polysaccharides (deacylated gellan gum or its salt, and acidic polysaccharides (e.g., alginic acid) or its salt that can maintain a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions) are loosened, and the three-dimensional network is partially destroyed, and it is expected that the recovery rate of cells or tissues will be improved.

[0105] The chelating agent is not particularly limited as long as it can form a complex with divalent metal cations such as calcium ions and magnesium ions (preferably calcium ions). For example, citric acid or its salt (e.g., trisodium citrate); EDTA or its salt (e.g., sodium edetate salts such as EDTA2Na, EDTA3Na, EDTA4Na, etc.); hydroxylethylethylenediaminetriacetate salts such as HEDTA3Na; EGTA or its salt; pentetate (diethylenetriaminepentaacetate); phytic acid; phosphonic acids such as etidronic acid and its salts such as sodium salts; sodium oxalate; polyamino acids such as polyaspartic acid and polyglutamic acid; sodium polyphosphate; sodium metaphosphate; phosphoric acid; alanine; dihydroxyethylglycine; gluconic acid; ascorbic acid; succinic acid; tartaric acid, etc. can be mentioned. From the viewpoint of improving the recovery rate of cells or tissues, citric acid or its salt (e.g., trisodium citrate) or EDTA or its salt (e.g., sodium edetate salts such as EDTA2Na, EDTA3Na, EDTA4Na, etc.) is used. or its salt (e.g., sodium edetate salts such as EDTA2Na, EDTA3Na, EDTA4Na, etc.) A chelating agent is preferably a chelating agent that can form a chelate with a metal cation, and more preferably a chelating agent that can form a chelate with a divalent metal cation. Two or more chelating agents can be mixed and used. The combinations of chelating agents are not particularly limited, and examples thereof include combinations of citric acid or a salt thereof (e.g., trisodium citrate) and EDTA or a salt thereof (e.g., sodium edetate salts such as EDTA2Na, EDTA3Na, and EDTA4Na).

[0106] The addition amount of the chelating agent is an amount that can loosen the bonds between polysaccharides (deacylated gellan gum or a salt thereof, and an acidic polysaccharide (e.g., alginic acid) or a salt thereof that maintains a random coil state in a divalent metal cation medium and can be cross-linked via divalent metal ions) in the three-dimensional network contained in the liquid composition of the present invention through metal cations in the three-dimensional network. For example, in the case of citric acid or a salt thereof (e.g., trisodium citrate), usually, the final concentration immediately after addition is 0.001 w / v% or more, preferably 0.005 w / v% or more. Theoretically, the upper limit is the saturation concentration of citric acid or a salt thereof, but if the concentration is too high, there is concern about the effect on the viability of cells or tissues. Therefore, it is usually 0.2 w / v% or less, more preferably 0.1 w / v% or less. In addition, in the case of EDTA or a salt thereof (e.g., sodium edetate salts such as EDTA2Na, EDTA3Na, and EDTA4Na) usually, the final concentration immediately after addition is 0.001 w / v% or more, preferably 0.005 w / v% or more. Theoretically, the upper limit is the saturation concentration of EDTA or a salt thereof, but if the concentration is too high, there is concern about the effect on the viability of cells or tissues. Therefore, it is usually 0.2 w / v% or less, more preferably 0.1 w / v% or less.

[0107] After adding a chelating agent to the preservation preparation, it is preferable to perform shear force on the above-mentioned preservation preparation so that the chelating agent becomes uniform, and to stir well.

[0108] After the above pretreatment step, the resulting mixture containing cells or tissues is subjected to centrifugation to precipitate the cells or tissues and remove fractions other than the cells or tissues (for example, the liquid composition of the present invention in the supernatant), whereby the cells or tissues can finally be recovered from the storage preparation of the cells or tissues. The technique of precipitating cells and tissues by centrifugation is well known to those skilled in the art, and those skilled in the art can set appropriate conditions according to the types of cells and tissues. Generally, by centrifuging at a centrifugal force of about 10 to 400 G, the cells and tissues can be precipitated and separated from the supernatant.

[0109] As described above, since the loss of the effect of maintaining the suspended state of cells and tissues due to shear forces such as pipetting and filter filtration is a reversible reaction, it is preferable to subject the mixture to centrifugation before the three-dimensional network (amorphous structure) is regenerated after the above pretreatment step. For example, centrifugation is started within 60 minutes, preferably within 30 minutes, more preferably within 10 minutes after completion of the above pretreatment step.

[0110] The present invention will be described in more detail by specifically describing examples of the liquid composition of the present invention below, but the present invention is not limited thereto.

Example

[0111] [Test Example 1] Preparation of polysaccharide mixture 1 part by mass of sodium alginate (ALG) (Kimica Alginate IL-2, manufactured by Kimica Corporation) and 99 parts by mass of purified water were added to a glass culture medium bottle, and autoclaved (121 ° C, 20 min) to prepare a 1% by mass concentration ALG aqueous solution. Similarly, a 1% by mass concentration deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) aqueous solution was prepared. The ALG aqueous solution and the DAG aqueous solution were aliquoted into a conical tube at a ratio of 2:1 (v / v), and a syringe ​Using a disposable syringe equipped with a needle, pipetting and mixing were carefully performed to homogenize the solution, and a polysaccharide mixture was prepared.

[0112] [Test Example 2] Preparation of Liquid Composition A medium preparation kit (Nissan Chemical Industries FCeM (R) -series Preparation Kit) was used to prepare the liquid composition of the present invention. A predetermined amount of various media was dispensed into a conical tube (Sumitomo Bakelite 50 mL centrifuge tube), and an adapter cap, which is a component of the kit, was attached. The tip of a disposable syringe filled with the polysaccharide mixture obtained in Test Example 1 in a predetermined amount was fitted into and connected to the cylindrical part of the adapter -cap. The plunger of the syringe was manually pressed, and the polysaccharide mixture in the syringe was vigorously injected into the container to be instantaneously mixed with the medium, thereby preparing the liquid composition of the present invention. The prepared liquid composition is shown in Table 1.

[0113]

Table 1

[0114] [Analysis] Viscosity Measurement of Liquid Composition The viscosity of the liquid composition prepared in Test Example 2 was measured. As a representative example, DHb087 was measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1°34´×R24) at 25 °C and a rotation speed of 100 rpm for 5 minutes. As a result, the average value of the three measured values was 2.07 mPa·s (1.95 mPa·s for the first measurement, 2.21 mPa·s for the second measurement, and 2.05 mPa·s for the third measurement).

[0115] [Test Example 3] Cell Preservation (Cell Type: NHDF) Normal human neonatal foreskin fibroblasts (NHDF, manufactured by Kurabo Industries Ltd.) were prepared as a cell suspension containing 152×10 4 cells, and 38×10 4 cells were dispensed into four conical tubes each. Centrifugation (300 x g ​, after performing washing and removal, 1.9 mL of a liquid composition (DHb086 to DHb090) containing DAG and ALG (total concentration 0.016 (w / v)%) shown in Table 1 or a liquid composition not containing DAG and ALG as a comparison (Comparative Example 1 or Comparative Example 2) was added and resuspended to prepare cell suspensions containing each liquid composition. (20×10 4 cells / mL) were prepared. 100 μL of each of these was dispensed into 18 round-bottom microtubes (1.5 mL each), the lids were closed, and they were stored statically at 25 (±1)°C. On each day of the start of storage, 1st day, 4th day, 7th day, 14th day, and 21st day after storage, 3 tubes each were taken out, and the amount of ATP contained in the cells was quantified using CellTiter-Glo reagent (manufactured by Promega) with a plate reader (infinite M200 PRO, manufactured by Tecan). Using the RLU value measured on the start day of storage as a reference (cell viability 100%), the RLU values measured after storage for each number of days were compared, and the cell viability after the passage of time was calculated in this way to evaluate cell storage stability. The average value of the three measurements made in the above test was shown in Table 2. From Table 2, in Comparative Example 1 which did not contain the polysaccharide composition, the viability decreased by up to 30%, whereas

[0116]

Table 2

[0117] in DHb086, a liquid composition containing DAG and ALG, about 70% survival was confirmed even after 3 weeks, and in DHb090, 50% survival was confirmed 4 days later. From this, it was confirmed that the liquid composition containing DAG and ALG exhibits a cell storage effect. [Test Example 4] Cell storage (cell type: NHDF)

[0118] Normal human neonatal foreskin fibroblasts (NHDF, manufactured by Kurabo Industries Ltd.) were prepared as a cell suspension containing 310×10 cells, and 62×10 4 cells were dispensed into 5 conical tubes. Centrifugation (300 x g 4 cells / mL) , 3 minutes) was performed to remove the supernatant, and then the DAG and ALG shown in Table 1 (total concentrations 0.010, 0.013, 0.016 , 0.020 (w / v)%) liquid compositions (Table 1 DHb084, DHb085, DHb086, or DHb087) or, for comparison, a liquid composition without DAG and ALG (Comparative Example 1) 3.1 mL was added and resuspended, and cell suspensions (20×10 cells / mL) containing each liquid composition were prepared. These were dispensed 100 μL per well into 5 wells each of a 96-well U-bottom 4 cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd., MS-309UR), covered with lids, and stored statically at 25 (±1)°C. The amount of ATP contained in the cells in each 5 wells was quantified using CellTiter-Glo reagent (manufactured by Promega) with a plate reader (infinite M200PRO, manufactured by Tecan) on each day of the start of storage, 1 day, 3 days, 7 days, 14 days, and 21 days after storage. Using the RLU value measured on the start day of storage as a reference (cell viability 100%), the RLU values measured after storage for each number of days were compared, and the cell viability after the passage of time was calculated to evaluate cell storage stability. The average value of the 5 measurements in the above test was recorded in Table 3. From Table 3, at any polysaccharide concentration, a survival rate of about 70% or more was shown in 3 weeks, and a high cell storage effect of the liquid composition containing DAG and ALG

[0119]

Table 3

[0120] was confirmed. From Table 3, at any polysaccharide concentration, a survival rate of about 70% or more was shown in 3 weeks, and a high cell storage effect of the liquid composition containing DAG and ALG was confirmed.

[0121] [Test Example 5] Cell storage (cell type: h-MSC) A cell suspension containing human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) at 104×10 4 cells was prepared, and 26×10 4Cells were dispensed one by one. After centrifugation (300 x g, 3 minutes) to remove the supernatant, a liquid composition containing DAG and ALG shown in Table 1 (total concentration 0.016 (w / v)%) substances (Examples DHb086 to DHb090), or as a comparison, a liquid composition not containing DAG and ALG (Comparative Examples 1 to 2) 1.3 mL was added and resuspended, and cell suspensions (20×10 4 cells / mL) containing each liquid composition were prepared. These were dispensed 100 μL each into 12 1.5 mL round-bottom microtubes, the lids were closed, and they were stored statically at 25 (±1)°C. On each day of the start of storage, the 3rd day after storage, and the 7th day after storage, 3 tubes each were taken out, and the amount of ATP contained in the cells was determined using CellTiter-Glo reagent (manufactured by Promega) with a plate reader (infinite M200PRO, manufactured by Tecan). Using the RLU value measured on the start day of storage as a reference (cell viability 100%), the RLU values measured after storage for each number of days were compared, and the cell viability after the passage of time was calculated to evaluate cell storage stability . The average value of the three measurements taken in the above test was recorded in Table 4.

[0122]

Table 4

[0123] From Table 4, in DHb086, about 70% survived even after one week, and in DHb090, about 60% survived. Since the liquid composition containing DAG and ALG showed about a two-fold improvement in survival rate, the excellent cell storage effect of the liquid composition containing DAG and ALG was confirmed.

[0124] [Test Example 6] Cell storage test with different cell seeding densities and storage temperatures (cell type: h-MSC) A cell suspension of 17 mL containing 510×10 4 human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) was prepared, and 3×10 4 cells (0.1 mL), 30×104 Cells (1 mL), 150×10 4 Cells (5 mL), 300×10 4 Cells (10 mL) were each aliquoted. Centrifugation (300 x g, 3 minutes) was performed, and after supernatant removal, a liquid composition (DHb086) containing DAG and ALG shown in Table 1 (total concentration 0.016 (w / v)%) was added and resuspended to prepare cell suspensions at each cell density (1×10 cells / mL, 10×10 4 cells / mL, 50×10 4 cells / mL, 100×10 4 cells / mL). These were each aliquoted 100 μL into 24 1.5-mL round-bottom microtubes, capped, and stored statically at 25 (±1)°C or 37°C. On the storage start date, 3 tubes each were taken out on the 1st, 3rd, and 7th days after storage, and the amount of ATP contained in the cells was quantified using the CellTiter-Glo reagent (manufactured by Promega) with a plate reader (infinite M200PRO, manufactured by Tecan 4 Corporation). Using the RLU value measured on the storage start date as a reference (cell viability 100%), the RLU values measured after storage for each number of days were compared, and the cell viability after the passage of time was calculated to evaluate cell storability. The average value of the three measurements made in the above test was recorded in Table 5. From Table 5, the liquid composition containing DAG and ALG showed excellent cell storability at any cell seeding density in the storage test at 25°C. On the other hand, in the storage test at 37°C, the viability decreased significantly, confirming its effectiveness for storage at room temperature.

[0125]

Table 5

[0126] From Table 5, the liquid composition containing DAG and ALG showed excellent cell storability at any cell seeding density in the storage test at 25°C. On the other hand, in the storage test at 37°C, the viability decreased significantly, confirming its effectiveness for storage at room temperature.

[0127] [Test Example 7] Preservation of Spheres (Cell Type: h-MSC) Human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) were at 32.4×10 4 ​​Prepare a cell suspension containing cells, and add 4 mL of the cell suspension containing 32.4×10 cells to one well of a 6-well plate for spheroid preparation (Elplasia Spheroid Generators MPc500, manufactured by Kuraray Co., Ltd.). Culture for 3 days in DMEM-Low Glucose medium containing serum under the conditions of 37°C and 5% carbon dioxide to prepare spheroids of h-MSCs. Since 650 pinholes with a diameter of 500 μm are opened on the bottom surface of the well, it is seeded at a density of 500 cells per bottom surface of the hanging drop-shaped pinhole, and about 650 spheroids formed in each pinhole are obtained. The obtained spheroids are collected in a 15 mL conical tube, centrifuged 4 (100 x g, 1 minute) to remove the supernatant, and then 1.3 mL of a liquid composition (DHb087) containing DAG and ALG shown in Table 1 (total concentration 0.020 (w / v )%) is added and resuspended to prepare a spheroid suspension (50 cells / 100 μL). These are dispensed 100 μL each into 12 1.5 mL round-bottom microtubes, the lids are closed, and they are stored statically at 25 (±1)°C. Three of each are taken out on each day of the start of storage, the 3rd day, the 5th day, and the 7th day after storage, and the amount of ATP contained in the cells is quantified using CellTiter-Glo reagent (manufactured by Promega Corporation) with a plate reader (infiniteM200PRO, manufactured by Tecan Group Ltd.). Using the RLU value obtained by measurement on the start day of storage as a reference (cell viability 100%), the RLU values obtained by measurement after storage for each number of days are compared, and the cell viability after the passage of time is calculated to evaluate the cell storage stability. The average value of the three measurements made in the above test is shown in Table 6. In addition, the appearance of the spheroid storage suspension after standing for 7 days and the observation photos of the spheroids before and after storage are shown in Figure 1. )%) is added and resuspended to prepare a spheroid suspension (50 cells / 100 μL). These are dispensed 100 μL each into 12 1.5 mL round-bottom microtubes, the lids are closed, and they are stored statically at 25 (±1)°C. Three of each are taken out on each day of the start of storage, the 3rd day, the 5th day, and the 7th day after storage, and the amount of ATP contained in the cells is quantified using CellTiter-Glo reagent (manufactured by Promega Corporation) with a plate reader (infiniteM200PRO, manufactured by Tecan Group Ltd.). Using the RLU value obtained by measurement on the start day of storage as a reference (cell viability 100%), the RLU values obtained by measurement after storage for each number of days are compared, and the cell viability after the passage of time is calculated to evaluate the cell storage stability. The average value of the three measurements made in the above test is shown in Table 6. In addition, the appearance of the spheroid storage suspension after standing for 7 days and the observation photos of the spheroids before and after storage are shown in Figure 1. test is shown in Table 6. In addition, the appearance of the spheroid storage suspension after standing for 7 days and the observation photos of the spheroids before and after storage are shown in Figure 1. The average value of the three measurements made in the above test is shown in Table 6. In addition, the appearance of the spheroid storage suspension after standing for 7 days and the observation photos of the spheroids before and after storage are shown in Figure 1. The average value of the three measurements made in the above test is shown in Table 6. In addition, the appearance of the spheroid storage suspension after standing for 7 days and the observation photos of the spheroids before and after storage are shown in Figure 1.

[0128] [Table 6]

[0129] From Table 6, it was confirmed that the spheres survived without dying even after 5 days of storage, and more than 80% survived even after 7 days. When stored in the medium of Comparative Example 1, the spheres aggregated with each other and were excluded from the test because necrosis occurred inside the aggregates (data not shown).

[0130] From Figure 1, it was confirmed that the morphology of the spheres was maintained before and after storage, and they remained floating in the liquid composition containing DAG and ALG even after 7 days of storage.

[0131] [Test Example 8] Storage of Spheres in a Vibration Environment (Cell Type: h-MSC) A cell suspension containing human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) at 64.8×10 4 cells was prepared, and 32.4×10 cells per well of the cell suspension were added to each of the 2 wells of a 6-well plate for sphere preparation (Elplasia Spheroid Generators MPc500, manufactured by Kuraray), and cultured in DMEM-Low Glucose medium containing serum for 3 days at 37°C and 5% carbon dioxide conditions to prepare h-MSC spheres. Since 650 pinholes with a diameter of 500 μm were opened on the bottom surface of the well, 4 seeding was performed at a density of 500 cells per bottom surface of the hanging drop-shaped pinholes, and approximately 650 spheres / well formed in each pinhole were obtained. The obtained spheres were collected into 15 mL conical tubes for each well, centrifuged (100 x g, 1 minute) to remove the supernatant, and then 1.3 mL of the liquid composition (DHb087) containing DAG and ALG (total concentration 0.020 (w / v)%) shown in Table 1 or a liquid composition without DAG and ALG (Comparative Example 1) as a comparison was added and resuspended, and each sphere was resuspended. The spheres formed in each pinhole were seeded at a density of 500 cells per bottom surface of the hanging drop-shaped pinholes, and approximately 650 spheres / well were obtained. The obtained spheres were collected into 15 mL conical tubes for each well, centrifuged (100 x g, 1 minute) to remove the supernatant, and then 1.3 mL of the liquid composition (DHb087) containing DAG and ALG (total concentration 0.020 (w / v)%) shown in Table 1 or a liquid composition without DAG and ALG (Comparative Example 1) as a comparison was added and resuspended, and each sphere A suspension (50 cells / 100 μL) was prepared. 100 μL of this suspension was dispensed into each of 12 1.5-mL round-bottom microtubes, the lids were closed, and they were stored at 25 (±1)°C in a vibrating environment (ASCM-1 high-speed shaker (equipped with a black tube for 1.5 mL, 300 rpm), manufactured by AS ONE Corporation). On each of the storage start day, the 3rd day after storage, and the 7th day after storage, 3 tubes were taken out each day, and the amount of ATP contained in the cells was quantified using CellTiter-Glo reagent (manufactured by Promega Corporation) with a plate reader (infinite M200 PRO, manufactured by Tecan Group, Ltd.). Using the RLU value measured on the storage start day as a reference (cell viability 100%), the RLU values measured after storage for each number of days were compared, and the cell viability after the passage of time was calculated to evaluate the cell storage stability. The average value of the three measurements made in the above test was shown in Table 7. The cell storage stability was evaluated by comparing the RLU values measured after storage for each number of days with the RLU value measured on the storage start day as a reference (cell viability 100%) and calculating the cell viability after the passage of time. The average value of the three measurements made in the above test was shown in Table 7. When a storage test of h-MSC spheres was carried out under vibration conditions assuming the environment during transportation, from Table 7, the liquid composition containing DAG and ALG showed good cell storage stability even under vibration conditions. In Comparative Example 1, although the aggregation of the spheres was slightly suppressed due to vibration, a decrease in viability was observed.

[0132] [Table 7]

[0133] When a storage test of h-MSC spheres was carried out under vibration conditions assuming the environment during transportation, from Table 7, the liquid composition containing DAG and ALG showed good cell storage stability even under vibration conditions. In Comparative Example 1, although the aggregation of the spheres was slightly suppressed due to vibration, a decrease in viability was observed.

[0134] [Reference Example 1] Preparation of polysaccharide mixture 1 part by mass or 2 parts by mass of sodium alginate (ALG) (KIMICA GIN, manufactured by KIMICA CORPORATION) and 99 parts by mass or 98 parts by mass of purified water were added to a glass culture medium bottle, and autoclave sterilization treatment (121°C, 20 minutes) was performed to prepare a 1% by mass concentration or 2% by mass concentration ALG aqueous solution. Similarly, 1% by mass concentration and 2% by mass concentration deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) aqueous solutions were prepared. Similarly, 1% by mass concentration and 2% by mass concentration deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sankyo Co., Ltd.) aqueous solutions were prepared. A given amount of the ALG aqueous solution and the DAG aqueous solution were dispensed into a microtube, and thoroughly mixed and homogenized by pipetting using a disposable syringe equipped with a syringe needle to prepare a polysaccharide mixture.

[0135] [Reference Example 2] Preparation of liquid composition (1) Preparation of liquid composition using a vortex mixer Add a specified amount of solution to a conical tube (Sumitomo Bakelite 15 mL, 50 mL, or 225 mL centrifuge tube) The medium was dispensed and stirred with a vortex mixer while left open. The polysaccharide mixture was vigorously injected into the medium from a disposable syringe (Terumo Terumo Syringe) equipped with a syringe needle (Fuchigami Kikai FN5200) filled with a predetermined amount of the polysaccharide mixture obtained in Reference Example 1. A liquid composition was prepared by adding

[0136] (2) Culture medium preparation kit (Nissan Chemical Industries FCeM TM -series Preparation Kit) Preparation of the composition A predetermined amount of medium was dispensed into a conical tube (Sumitomo Bakelite 50 mL centrifuge tube), and an adapter cap, which is a component of the kit, was attached. A predetermined amount of the polysaccharide mixture obtained in Reference Example 1 was filled. The tip of the loaded disposable syringe was fitted into the cylindrical portion of the adapter cap to connect it, and the plunger of the syringe was manually pressed, forcibly ejecting the polysaccharide mixture in the syringe into the container and bringing it into contact with the culture medium to produce a liquid composition.

[0137] [Reference Example 3] Confirmation of floating effect In the liquid composition prepared in Reference Example 2, a polymer was added to simulate floating cells. Add polyethylene beads (diameter 500-600 μm, manufactured by Polysciences Inc.) and stir, then stop stirring. Ten minutes after the injection, the dispersion state of the beads in the liquid was visually confirmed. 2+An adequate amount of the structure formed by cross-linking through (etc.) is appropriately dispersed in the liquid in a finely divided state, the beads also remain dispersed and floating in the liquid. On the other hand, if the dispersion of the structure is not sufficient, the beads will accordingly settle. Regarding the dispersion state of the beads, a preferably dispersed and floating state is represented by ○, a state where they are dispersed but some have settled is represented by △, and a state where all the beads have settled is represented by ×.

[0138] (1) Floating effect when using DAG and ALG (1:1)

[0139]

Table 8

[0140] (2) Floating effect when using DAG and ALG (0.5:1)

[0141]

Table 9

[0142] [Reference Example 4] Cell recovery by pipetting Normal human neonatal foreskin fibroblasts (NHDF, Kurabo Industries) in the logarithmic growth phase were prepared at 1800×10 4 cells, and dispensed at 300×10 4 cells each. After centrifugation (300 x g, 3 minutes) to remove the supernatant, 30 mL of a liquid composition containing DAG and ALG (total concentration 0.015 (w / v)%) in various ratios (Examples C369 to C373 in Table 10) was added and gently stirred to prepare a cell suspension (10×10 4 cells / mL). 10×10 4The cell suspension of cell division was added at 1 mL per well and cultured for 1 week under the conditions of 37°C and 5% carbon dioxide. After measuring the cell concentration of the cell suspension after culture using a cell counter (TC-20, BIO-RAD), the suspension was transferred to a 1.5 mL microtube and homogenized by pipetting 20 times with a micropipette (manufactured by Thermo Scientific, ClipTip 1000 μL) with a suction and discharge volume set to 0.2 mL. Then, centrifugation (300 x g, 3 minutes) was performed, 1.1 mL of the supernatant was removed, 0.9 mL of DMEM-LG containing 10% fetal bovine serum was added and resuspended, and the amount of ATP contained in the cells was quantified using CellTiter-Glo (Promega) with a plate reader (manufactured by Tecan). Using the RLU value obtained by measuring the cell suspension after culture before the cell recovery operation as a reference (cell recovery rate 100%), the cell recovery rate was calculated by comparing the RLU value obtained when a floating inhibitor was added and the cell recovery operation was performed. All of the above tests were carried out 3 times, and the average value was recorded in a table .

[0143] [Table 10]

[0144] [Reference Example 5] Examination of the number of pipetting times Normal human neonatal foreskin fibroblasts (NHDF, Kurashiki Boseki) in the logarithmic growth phase were prepared at 1440×10 4 cells, suspended in 48 mL of the liquid composition of Example C371 in Table 10, and dispensed at 30×10 4 cells (1 mL) each into a 24-well cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd.) and cultured for 3 days under the conditions of 37°C and 5% carbon dioxide. After measuring the cell concentration of the cell suspension after culture using a cell counter (TC-20, BIO-RAD), the suspension was transferred to a 1.5 mL microtube and a micropipette (manufactured by Thermo Scientific, ClipTip It was pipetted a predetermined number of times with a micropipette (Thermo Scientific, ClipTip 1000 μL). Then, centrifugation (300 x g, 3 minutes) was performed. Next, 1.1 mL of the supernatant was removed, 0.9 mL of DMEM-LG containing 10% fetal bovine serum was added and resuspended. Then, the amount of ATP contained in the cells was quantified using CellTiter-Glo (Promega) with a plate reader (Tecan), and the cell recovery rate was calculated. All of the above tests were performed 5 times, and the average value was recorded in the table.

[0145]

Table 11

[0146] [Reference Example 6] Addition of Chelating Agent Normal human neonatal foreskin fibroblasts (NHDF, Kurabo) in the logarithmic growth phase were prepared at 450×10 4 cells and suspended in 15 mL of the liquid composition of Example C371 in Table 10. 30×10 4 cells (1 mL each) were dispensed into a 24-well cell culture plate (manufactured by Sumitomo Bakelite), and cultured under conditions of 37°C and 5% carbon dioxide for 3 days. After measuring the cell concentration of the cell suspension after culture using a cell counter (TC-20, BIO-RAD), the suspension was transferred to a 1.5 mL microtube, and a predetermined amount of chelating agent (mixed aqueous solution of 0.033 (w / v)% EDTA-2Na and 0.007 (w / v)% sodium citrate) in an amount of 0 to 0.1 mL was added, and pipetting was performed 0 or 10 times with a micropipette (manufactured by Thermo Scientific, ClipTip 1000 μL) with a suction / discharge volume set to 0.2 mL. Then, centrifugation (300 x g, 3 minutes) was performed, 1.1 mL of the supernatant was removed, 0.9 mL of DMEM-LG containing 10% fetal bovine serum was added and resuspended. Then, the amount of ATP contained in the cells was measured using CellTiter-Glo Quantification was performed using a plate reader (manufactured by Tecan) with (Promega), and the cell recovery rate was calculated. All of the above tests were performed three times, and the average values were recorded in a table.

[0147]

Table 12

[0148] [Test Example 7] Proliferation of Jurkat cells Human T-cell leukemia-derived cells (Jurkat E6.1, DS Pharma Biomedical Co., Ltd.) in the logarithmic growth phase were prepared at 240×10 4 cells, and centrifuged at 40×10 4 cells each (300 x g, 3 minutes) After removing the supernatant, a liquid composition containing DAG and ALG (mass ratio 1:0.5) at various concentrations (Examples DHb020 to DHb023 in Table 13 ), and a liquid composition containing DAG and not containing ALG (Comparative Example DHb024 in Table 13) were added at 8 mL and gently stirred to prepare a cell suspension (5×10 4 cells / mL). A 96-well U-bottom cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd., MS-309UR) was added with 0.1 mL of the cell suspension per well at 0.5×10 4 cells, and cultured at 37°C under 5% carbon dioxide conditions for 1 or 4 days The number of cells before and after culture was compared using a plate reader (manufactured by Tecan, infiniteM200PRO) with CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) as the amount of ATP contained in the cells. All of the above tests were performed four times, and the average values were recorded in a table.

[0149]

Table 13

[0150] As a result of the evaluation, even when the liquid composition of the present invention was used, cell growth equivalent to that of the comparative example was achieved.

[0151] [Reference Example 8] A549 Cell Proliferation Human alveolar basal epithelial adenocarcinoma cells (A549, DS Pharma Biomedical Co., Ltd.) in the logarithmic growth phase were prepared at 86.4×10 4 cells. After centrifugation (300 x g, 3 minutes) to remove the supernatant, a liquid composition containing DAG and ALG (mass ratio 1:0.5) at various concentrations (Examples E041, E045, E047, E048 in Table 14), and a liquid composition containing DAG and not containing ALG (Comparative Example E049 in Table 14) were added at 8 mL and gently stirred to prepare a cell suspension (5×10 4 cells / mL). A 96-well U-bottom cell culture plate (manufactured by Sumitomo Bakelite Co., Ltd., MS-309UR) was added with 0.5×10 4 cells of the cell suspension per well or 0.1 mL each, and cultured at 37°C under 5% carbon dioxide conditions for 1 or 4 days. The number of cells before and after culture was compared using a plate reader (manufactured by Tecan, infinite M200PRO) with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) as the amount of ATP contained in the cells. All of the above tests were performed 6 times, and the average values were recorded in a table. As a result of the evaluation, even when the liquid composition of the present invention was used, cell growth equivalent to that of the comparative example was achieved.

[0152]

Table 14

[0153] As a result of the evaluation, even when the liquid composition of the present invention was used, cell growth equivalent to that of the comparative example was achieved.

[0154] [Reference Example 9] Cell Recovery on a 10 mL Scale A549 cells were seeded into 200 mL of the liquid composition of Example E041 in Table 14 to a concentration of 10×10 4 cells / mL, and cultured at 37°C under 5% carbon dioxide conditions for two days. After culture, the cell suspension was divided into 10 mL portions The cells were collected, 1 mL of a chelating agent (a mixed aqueous solution of 0.033 (w / v)% EDTA-2Na and 0.007 (w / v)% sodium citrate) was added, and the cells were immediately strained using cell strainers (40 μm, 70 μm, 100 μm, Falcon®). The cells were then passed through a centrifugation strainer under various conditions (50×g, 100×g, 300×g, g: gravity acceleration) for 3 minutes. The number of cells before and after the cell recovery procedure was counted as the amount of ATP contained in the cells using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571). The results were compared using a plate reader (Tecan, infiniteM200PRO).

[0155] [Table 15]

[0156] As a result of the evaluation, it was shown that the liquid composition of the present invention can achieve a high cell recovery rate by passing the cells through a mesh (cell strainer) instead of pipetting. [Industrial Applicability]

[0157] By using the liquid composition of the present invention, cells or tissues can be stored for a long period of time, for example, at room temperature, in an unfrozen state, while maintaining good viability. When spheres are stored in the liquid composition of the present invention, aggregation of the spheres is avoided, and the occurrence of necrosis inside the spheres is suppressed.

[0158] The contents of all publications, including patents, patent applications, and scientific literature mentioned herein are hereby incorporated by reference to the same extent as if fully set forth herein.

[0159] This application is based on patent application No. 2017-173479 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

【Claim 1】 The invention described in the specification.

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