Cell preservation materials

Abstract

An objective of the present invention is to provide a technique for preserving cells or tissues in an unfrozen state while maintaining good viability. Cells or tissues are preserved in an unfrozen state in a liquid composition containing 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 acidic polysaccharide may be alginic acid. The liquid composition may further contain a metal cation such as calcium ion.

Description

[Technical Field]

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

[0002] Currently, in transplantation medicine research using stem cells such as iPS cells and mesenchymal stem cells, cell banks prepare large quantities of stem cells and cryopreserve them. Research institutions and medical institutions order the desired stem cells from the cell bank stock in a frozen state, thaw them, induce dormancy, and differentiate them into the desired cells as needed for various applications. However, in the cell bank system that operates an autologous transplant system and manages and stores a large number of allogeneic cell stocks, current transportation technologies that require freezing cells make it difficult to provide a large and stable supply of cells in a state suitable for transplantation. Specifically, for transplantation, cryopreserved cells must be thawed, cultured, and differentiated into the desired cells as needed to provide a large number of cells in good condition. However, if cells must be transported frozen, transplantation facilities must order frozen cells from the cell bank that stores and manages them and perform mass culture in-house. Therefore, transplantation facilities are required to have mass cell culture facilities (e.g., cell processing centers (CPCs)), and facilities without such facilities find it difficult to perform transplantation therapy. On the other hand, cell banks typically have the technology and equipment for mass cell culture. Therefore, if the cell bank can prepare large quantities of cells in a good condition suitable for transplantation and quickly supply the obtained cells to transplantation facilities while maintaining their good condition, transplantation therapy can be performed even at facilities that do not have mass cell culture facilities. To solve this problem, technology is essential for preserving and transporting large quantities of cells while maintaining their good condition without freezing.

[0003] Polysaccharides such as deacylated gellan gum (DAG) form three-dimensional networks (amorphous structures) in water by assembling through metal cations (e.g., divalent metal cations such as calcium ions). When cells are cultured in a liquid medium containing this three-dimensional network, the cells are trapped in the three-dimensional network and do not sink. This allows the cells to be cultured in a uniformly dispersed suspended state (suspension static culture) without the need for shaking, rotation, or other manipulations. Furthermore, because the above-mentioned three-dimensional network can be formed without substantially increasing the viscosity of the liquid medium, medium compositions containing this three-dimensional network also have excellent operability in subcultures and other processes (Patent Document 1). This medium composition, which is suitable for suspension static culture, has various excellent properties, such as enhancing the proliferation activity of various cells, and is therefore expected to be applied to a wide range of technical fields, such as regenerative medicine and mass production of proteins.

[0004] Patent Document 2 discloses that a medium composition containing nanofibers is used for preserving and transporting cells and tissues. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 017513 [Patent Document 2] International Publication No. 2015 / 111686 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a technique for preserving cells or tissues in an unfrozen state while maintaining good viability. [Means for solving the problem]

[0007] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that cells and tissues can be stored at room temperature for long periods of time while maintaining good viability by suspending them in a medium composition containing deacylated gellan gum and alginic acid. Storing spheres in this medium composition prevented aggregation of the spheres and suppressed the occurrence of necrosis within the spheres. Cells could be stored in this medium composition for long periods of time while maintaining good viability at room temperature, even under vibration conditions simulating the environment during transportation. Based on these findings, the present inventors conducted further research and completed the present invention.

[0008] That is, the present invention is as follows: [1] A liquid composition for preserving cells or tissues in an unfrozen 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 liquid composition according to [1], wherein the concentration of the deacylated gellan gum or a salt thereof in the liquid composition is 0.002 to 0.01 (w / v)% in terms of free form 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 the deacylated gellan gum or a salt thereof is 1 or more in terms of free form. [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 comprising a metal cation. [6] The liquid composition according to [5], wherein the metal cation is a 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. [8] A method for preserving cells or tissues, comprising preserving the cells or tissues in an unfrozen state in the liquid composition according to any one of [1] to [7]. [9] The method described in [8], wherein the cells or tissues are preserved in a suspended state in the liquid composition.

[10] The method according to [8] or [9], wherein the cells or tissues are stored 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.

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

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

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

[12] , which preserves cells in a sphere state. [Effects of the Invention]

[0009] 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. Storing spheres in the liquid composition of the present invention prevents aggregation of the spheres and suppresses the occurrence of necrosis within the spheres.

[0010] The liquid composition of the present invention is also useful for transporting cells and tissues in an unfrozen state. For example, when cells are cultured on a plate and transported as is, vibrations during transport can cause the cells to detach from the plate, resulting in a decrease in the original functions of the cells. However, the liquid composition of the present invention can maintain the cells or tissues in a floating state, thereby preventing damage to the cells caused by detachment from the plate due to vibrations during transport and allowing the cells or tissues to be preserved and transported while maintaining their original functions. [Brief explanation of the drawings]

[0011] [Figure 1] The morphology of spheres before and after storage is shown. The left image shows the morphology of spheres at the start of storage. The center image shows the morphology of spheres after 7 days of storage. The right image shows spheres that remained suspended after 7 days of storage. DETAILED DESCRIPTION OF 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 an unfrozen state, which allows cells or tissues to be preserved in an unfrozen state while maintaining their viability.

[0014] In the present invention, a cell is the most basic unit constituting an animal or plant, and has as its elements a cytoplasm and various organelles within the cell membrane. In this case, the nucleus containing DNA may or may not be contained within the cell. For example, animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells that constitute an organism, stem cells, progenitor cells, cancer cells isolated from an organism, cells isolated from an organism that have acquired immortalization and are stably maintained ex vivo (cell lines), cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have undergone artificial nucleus replacement. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, and mononuclear cells. 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 neural tissue. Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Progenitor cells are cells that are in the process of differentiating from stem cells into specific somatic or germ cells. Cancer cells are cells that derive from somatic cells and have the ability to proliferate indefinitely.A cell line is a cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a living body. Examples of cell lines include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), and Vero.

[0015] Plant-derived cells in the present invention include cells isolated from various tissues of a plant body, and also include protoplasts obtained by artificially removing the cell wall from such cells.

[0016] In the present invention, a tissue is a structural unit in which cells with several different properties and functions are assembled in a certain pattern, and examples of animal tissues include epithelial tissue, connective tissue, muscle tissue, nervous tissue, etc. Examples of plant tissues include meristematic tissue, epidermal tissue, assimilated tissue, mesophyll tissue, conductive tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell mass (callus), etc.

[0017] The cells or tissues to be preserved in the liquid composition of the present invention can be selected from any of the cells or tissues described above. The cells or tissues can be directly collected from animals or plants. The cells or tissues can also be harvested after being derived, grown, or transformed from animals or plants through specific treatment. In this case, the treatment can be performed in vivo or ex vivo. Examples of animals include fish, amphibians, reptiles, birds, pancrustaceans, hexapods, and mammals. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, rhesus monkeys, chimpanzees, and humans. There are no particular limitations on plants, as long as the harvested cells or tissues can be cultured in liquid. Examples include, but are not limited to, plants (e.g., ginseng, periwinkle, henbane, coptis, belladonna, etc.) that produce herbal medicines (e.g., saponin, alkaloids, berberine, scopolin, plant sterols, etc.), plants (e.g., blueberry, safflower, madder, saffron, etc.) that produce pigments and polysaccharides (e.g., anthocyanins, safflower pigments, madder pigments, saffron pigments, flavones, etc.) that are used as ingredients in cosmetics and foods, and plants that produce pharmaceutical ingredients.

[0018] In a preferred embodiment, the liquid composition of the present invention allows for the preservation of living cells or tissues in an unfrozen state while maintaining the cells or tissues in a suspended state.

[0019] In the present invention, "suspension of cells or tissues" refers to a state in which the cells or tissues are not adhered (non-adherent) to a storage or culture vessel. Furthermore, in the present invention, when preserving cells or tissues in a liquid composition, a state in which the cells or tissues are uniformly dispersed and suspended in the liquid composition without applying external pressure or vibration to the liquid composition, or without shaking or rotating the liquid composition, is referred to as "suspended suspension," and preserving cells or tissues in this state is referred to as "suspended suspension preservation." Furthermore, the period during which cells or tissues can be suspended in "suspended suspension" includes 5 minutes or more (e.g., at least 5 to 60 minutes), 1 hour or more (e.g., 1 hour to 24 hours), 24 hours or more (e.g., 1 day to 21 days), 48 hours or more, 7 days or more, etc., but is not limited to these periods as long as the suspended state is maintained.

[0020] In a preferred embodiment, the liquid composition of the present invention allows cells or tissues to be suspended and kept still at at least one temperature within a temperature range (e.g., 0 to 37°C) that allows cells or tissues to be preserved in an unfrozen state. The liquid composition of the present invention allows cells or tissues to be suspended and kept still at at least one temperature within a temperature range of preferably 1 to 30°C, more preferably at least one temperature within a temperature range of 15 to 30°C, even more preferably at least one temperature within a temperature range of 22 to 28°C, still more preferably at least one temperature within a temperature range of 24 to 26°C, and most preferably at least 25°C.

[0021] Whether or not the cells to be preserved can be suspended can be determined by, for example, 4 The cells are uniformly dispersed in the liquid composition to be evaluated at a concentration of 10 ...

[0022] The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and is crosslinkable via divalent metal ions. The liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and is crosslinkable via divalent metal ions, thereby enabling cells or tissues to be preserved in an unfrozen state while maintaining good viability. In a preferred embodiment, the liquid composition of the present invention comprises deacylated gellan gum or a salt thereof, and an acidic polysaccharide or a salt thereof that maintains a random coil state in a divalent metal cation medium and is crosslinkable via divalent metal ions, thereby providing the liquid composition with a property that enables cells or tissues to be preserved in a suspended state (preferably, suspended static preservation) (the effect of maintaining cells or tissues in a suspended state).

[0023] Deacylated gellan gum is a linear polymeric polysaccharide whose constituent units are four sugar molecules: 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 greater). R1 ​​may contain a glyceryl group and R2 may contain an acetyl group, but the content of acetyl groups and glyceryl groups is preferably 10% or less, more preferably 1% or less.

[0024] [ka]

[0025] Deacylated gellan gum can be produced by culturing gellan gum-producing microorganisms in a fermentation medium, treating the extracellular mucus with alkali, deacylating the glyceryl and acetyl groups attached to 1-3-linked glucose residues, recovering the mucus, drying, grinding, and then powdering it. Purification methods include liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20 or similar, adsorption chromatography using activated carbon or silica gel or thin-layer chromatography to adsorb and desorb active substances, and high-performance liquid chromatography using a reversed-phase column, either singly or in any combination or repetition. Examples of microorganisms that produce gellan gum include, but are not limited to, Sphingomonas elodea and genetically modified versions of Sphingomonas elodea.

[0026] The deacylated gellan gum may also be phosphorylated, and the phosphorylation can be carried out by known techniques.

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

[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, and the like; 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; and salts with amino acids such as glycine, alanine, and valine.

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

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

[0031] Examples of acidic polysaccharides or salts thereof that maintain a random coil state in a medium containing 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) and can be crosslinked via a divalent metal ion include alginic acid, pectin, pectinic acid, and salts thereof, with alginic acid or a salt thereof being preferred.

[0032] Alginic acid is a polysaccharide having a linear polymer structure of both uronic acids, α1-4 linked L-glucuronic acid and β1-4 linked D-mannuronic acid.

[0033] Alginic acid or its salts can be extracted and purified from brown algae, such as kelp and wakame, by performing an ion exchange reaction with the carboxyl groups of alginic acid. Alginic acid in the algae forms insoluble salts with multivalent cations such as calcium ions, and this salt is then ion-exchanged with Na to form water-soluble sodium alginate, which can then be extracted from the algae. Furthermore, by adding acid to an aqueous solution of sodium alginate, the insoluble alginic acid is coagulated and precipitated, and the coagulated and precipitated alginic acid can be isolated to obtain purified alginic acid.

[0034] 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, and iron; 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; and salts with amino acids such as glycine, alanine, and valine. 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 a salt thereof is preferably 300 to 50,000,000, more preferably 500 to 10,000,000, and even more preferably 1,000 to 5,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).

[0036] As the alginic acid or a salt thereof, commercially available products such as the following products can also be used. Kimika Co., Ltd.: Kimika 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 Argitex Series LL, L, M, H Kikkoman Biochemifa Corporation: Duck alginate NSPH2R, NSPHR, NSPMR, NSPLR, NSPLLR Sansho Corporation: Scoggin, San Arguin Hokkaido Mitsui Chemicals, Inc.: Alginic acid oligosaccharide ALGIN

[0037] Deacylated gellan gum and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions may exist in the form of a tautomer, a geometric isomer, a mixture of tautomers or geometric isomers, or a mixture thereof, formed by endocyclic or exocyclic isomerization.Deacylated gellan gum and acidic polysaccharides that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions may exist in the form of resolved optical isomers or a mixture containing them in any ratio, if they have an asymmetric center, whether formed by isomerization or not.

[0038] 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, aggregate via metal cations (e.g., divalent metal cations such as calcium ions) in the liquid composition to form a three-dimensional network (irregular structure). It is known that polysaccharides form microgels via metal cations (e.g., JP 2004-129596 A), and one embodiment of the irregular structure includes such microgels. One embodiment of the metal cation-mediated aggregation of 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, is a film-like structure. The liquid composition of the present invention comprises a three-dimensional network (irregular structure) formed by the aggregation of this 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 is crosslinkable via divalent metal ions (e.g., divalent metal cations such as calcium ions) via metal cations. 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 settle, making it possible to store the cells or tissues in a uniformly dispersed suspended state (suspended and static storage) without the need for shaking, rotation, or the like. The liquid composition of the present invention preferably comprises the three-dimensional network (irregular structure) in a uniformly dispersed form.

[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. In this case, the viscosity of the liquid composition at 25°C is 8 mPa s or less, 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 Examples below. Specifically, it can be measured at 25°C using an E-type viscometer (TV-22 type viscometer, model: TVE-22L, manufactured by Toki Sangyo Co., Ltd., cone rotor: standard rotor 1°34' x R24, rotation speed 100 rpm).

[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, but examples thereof include polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides having sulfate or phosphate groups in part of their structure, or polysaccharides having both structures, and include not only naturally occurring polysaccharides but also polysaccharides produced by microorganisms, polysaccharides produced by genetic engineering, and polysaccharides artificially synthesized using enzymes. More specific examples include hyaluronic acid, native gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, or salts thereof.

[0042] The concentration of deacylated gellan gum or a salt thereof in the liquid composition of the present invention (equivalent to free deacylated gellan gum) 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)%.

[0043] The concentration (free form equivalent) 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 is capable of crosslinking 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)%, even more preferably 0.005 to 0.015 (w / v)%, and still more preferably 0.0066 to 0.0133 (w / v)%.

[0044] The concentration of deacylated gellan gum or a salt thereof is preferably 0.002 (w / v)% or more, preferably 0.003 (w / v)% or more, from the viewpoint of ensuring sufficient cell or tissue suspension. On the other hand, if this concentration is too high, the suspension effect becomes strong, which may result in a decrease in cell recovery rate or reduced handleability of the medium itself. Therefore, it is preferable to set the concentration to 0.01 (w / v)% or less, preferably 0.009 (w / v)% or less. The concentration of acidic polysaccharides (e.g., alginic acid) or salts thereof that maintain a random coil state in a divalent metal cation medium and can be crosslinked 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 of rapidly losing the effect of maintaining the suspension of cells or tissues due to shear force (vulnerability of the effect of maintaining the suspension of cells or tissues to shear force). On the other hand, if this concentration is too high, there is a risk of gelation, so it is preferable to keep the concentration at 0.1 (w / v)% or less, preferably 0.02 (w / v)% or less, and 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 maintains a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions, contained in the liquid composition of the present invention, is 1 part by mass or more, preferably 2 parts by mass or more, of the 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, per 1 part by mass of deacylated gellan gum or a salt thereof, from the viewpoint of achieving the property of rapidly eliminating the effect of maintaining the suspension of cells or tissues by shear force. In one embodiment, the mass ratio of the 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, per 1 part by mass of deacylated gellan gum or a salt thereof, is, for example, 1 to 4 parts by mass, preferably 1 to 3 parts by mass, and more preferably 1 to 2 parts by mass.

[0046] The compound concentration in the liquid composition can be calculated using 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 in the above-mentioned amounts, 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 is capable of crosslinking via divalent metal ions, thereby achieving the effect of maintaining good viability of cells or tissues preserved in an unfrozen state.The liquid composition of the present invention contains deacylated gellan gum or a salt thereof in the above-mentioned amounts, 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 is capable of crosslinking via divalent metal ions, thereby achieving the effect of maintaining a floating state of cells or tissues.

[0049] The liquid composition of the present invention contains deacylated gellan gum or a salt thereof in the above-mentioned amounts, 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, and therefore has the property that the effect of maintaining a suspended state of cells or tissues is quickly lost by shear forces such as pipetting or filtration (vulnerability of the effect of maintaining a suspended state of cells or tissues to shear forces).

[0050] The liquid composition of the present invention contains a three-dimensional network (irregular 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, via metal cations (e.g., divalent metal cations such as calcium ions), and this has the effect of maintaining cells and tissues in a suspended state. However, 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 makes the three-dimensional network vulnerable to chelating agents or shear forces, and this three-dimensional network is easily destroyed by shear forces such as pipetting or filtration, and the effect of maintaining cells and tissues in a suspended state is quickly lost. Deacylated gellan gum has structural units with a relatively linear structure, and multiple deacylated gellan gum chains bundle together in a liquid composition to form a tight and stable three-dimensional network. This three-dimensional network is therefore resistant to disruption by chelating agents, pipetting, filter filtration, and the like. However, when an acidic polysaccharide (e.g., alginic acid) or a salt thereof is added to a liquid composition, the bundling of deacylated gellan gum is inhibited, and the three-dimensional network becomes vulnerable to shear forces such as pipetting and filter filtration. This is because the uronic acids contained in the polysaccharides are both α1-4 linked L-glucuronic acid and β1-4 linked D-mannuronic acid, and therefore maintain a random coil state in a divalent metal cation medium, and can be crosslinked via divalent metal ions. However, this theory is not intended to be particularly binding.

[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 crosslinked 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 irregular structure). Therefore, the liquid composition of the present invention contains a metal cation, such as a divalent metal cation (e.g., calcium ion, magnesium ion, zinc ion, iron ion, or copper ion), preferably calcium ion. Two or more types of metal cations can be used in combination, such as calcium ion and magnesium ion, calcium ion and zinc ion, calcium ion and iron ion, or calcium ion and copper ion. Those skilled in the art can determine the appropriate combination. The metal cation concentration in the liquid composition of the present invention is 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM, but is not limited thereto.

[0052] The destruction of the three-dimensional network (loss of the ability to maintain the suspension of cells and tissues) due to shear forces such as pipetting and filtration is a reversible reaction because the fragments of the three-dimensional network (amorphous structure) destroyed by shear forces reassemble through metal cations (e.g., divalent metal cations such as calcium ions), allowing the three-dimensional network (amorphous structure) to be regenerated.

[0053] The liquid composition of the present invention preferably contains a medium (preferably a liquid medium) used to culture the cells or tissues to be preserved. In this case, the liquid composition of the present invention can be prepared by mixing the medium (preferably a liquid medium) used to culture the cells or tissues to be preserved with 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] Media used in culturing cells or tissues derived from animals (e.g., mammals) include, for example, Leibovitz's L-15 Medium, Dulbecco's Modified Eagle'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), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, and NutriStem MSC medium. XF (Biological Industries), NutriStem hPSC XF (Biological Industries), StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan Examples of such media include H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biologicals), StemPro hESCSFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), HFDM-1 (manufactured by Nipro), Nipro EIDF (manufactured by Nipro), and BMPro (manufactured by Nipro).

[0055] When cells or tissues are of plant origin, suitable media include basal media commonly used in plant tissue culture, such as Murashige-Skoog (MS) medium, Linsmeyer-Skoog (LS) medium, White's medium, Gamborg B5 medium, Nitsche's medium, Heller's medium, and Morel's medium, as well as modified media containing these media components at optimal concentrations (e.g., halving the ammonia nitrogen concentration) supplemented with appropriate concentrations of auxins and, if necessary, plant growth regulators (plant hormones) such as cytokinins. These media can be supplemented with caseinase, corn steep liquor, vitamins, etc., as needed. Examples of auxins include, but are not limited to, 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D). 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] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, and the like to the above-mentioned medium depending on the purpose. When culturing cells or tissues of animal origin, those skilled in the art may also add one or more combinations of other chemical or biological components depending on the purpose. Components that may be added to culture media for cells and / or tissues of animal origin include fetal bovine serum, human serum, horse 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, and various cell adhesion molecules. 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-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-29), interleukin-31 (IL-29), interleukin-32 (IL-29), interleukin-33 (IL-29), interleukin-34 (IL-29), interleukin-35 (IL-29), interleukin-36 (IL-29), interleukin-37 (IL-29), interleukin-38 (IL-29), interleukin-39 (IL-39), interleukin-40 (IL-39), These include, but are not limited to, interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), and thrombopoietin (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, Examples of hormones that may be involved include, but are not limited to, 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, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[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 inhibitory factor (PI), and phospholipase A (PPAR). These include, but are not limited to, phospholipase 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), and pleiotrophin.

[0059] Furthermore, it is also possible to add cytokines and growth factors whose amino acid sequences have been artificially modified using genetic engineering, such as the IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0060] Examples of various extracellular matrices and various cell adhesion molecules include collagens I to XIX, fibronectin, vitronectin, laminins 1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, hyaluronic acid, alginate gel, various hydrogels, and cleaved fragments thereof.

[0061] Examples of antibiotics added to the medium include sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexlocillin, mecillinam, andinocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enolic acid, and the like. These include fluoxacin, pipemidic acid, sulbactam, clavulic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, adinocillin and the formaldehyde foudate ester of sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, 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 (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), preferably calcium ions). When mixed with the liquid medium, the deacylated gellan gum or a salt thereof, and the 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 the metal cations (e.g., divalent metal cations such as calcium ions) in the liquid medium to form a three-dimensional network (an irregular structure). The concentration of metal cations (preferably calcium ions) in the medium is not particularly limited, as long as it is a concentration sufficient to allow 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 via the metal cations and form a three-dimensional network (irregular structure), but is, for example, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. A liquid medium containing the metal cation may be mixed with 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; alternatively, a medium not containing the metal cation may be mixed with 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, and then an aqueous solution containing the metal cation that has been separately prepared may be added to the mixture.

[0063] In addition to the above components, the liquid composition of the present invention may contain various components that have a cell survival-prolonging effect when cells or tissues are preserved in an unfrozen state. Examples of such components include, but are not limited to, sugars (excluding polysaccharides) (e.g., monosaccharides (e.g., 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. In one embodiment, the liquid composition of the present invention contains at least one compound selected from the group consisting of sugars (excluding polysaccharides) (e.g., monosaccharides (e.g., 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.

[0064] Furthermore, 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, thereby maintaining good viability of cells or tissues preserved in an unfrozen state. Therefore, it is not necessary to include other components that have a cell survival-prolonging effect when cells or tissues are preserved in an unfrozen state. 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 (e.g., 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.

[0065] The liquid composition of the present invention is intended for preserving cells or tissues in an unfrozen state and may therefore not contain a cryoprotectant. Examples of cryoprotectants include DMSO, glycerol, ethylene glycol, trimethylene glycol, methanol, dimethylacetamide, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, albumin, etc. In one embodiment, 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 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, are added to the liquid medium, the deacylated gellan gum or a salt thereof, and the 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 are first dissolved or dispersed in an appropriate solvent (this is used as a medium additive). Then, the medium additive can be added to the liquid medium so that the final concentrations of deacylated gellan gum or a salt thereof, and the 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 are as described 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 crosslinked via divalent metal ions may be prepared separately and 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 crosslinked 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 crosslinked 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 crosslinked 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 crosslinked via divalent metal ions) is prepared and added to a liquid medium.

[0067] Examples of suitable solvents for use in preparing the medium additive include, but are not limited to, hydrophilic solvents such as aqueous solvents such as water, physiological saline, and PBS, and various alcohols such as dimethyl sulfoxide (DMSO), methanol, ethanol, butanol, propanol, glycerin, propylene glycol, and butylene glycol. In this case, the concentrations of the deacylated gellan gum or a salt thereof and the 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 medium additive are desirably, for example, 10 to 500 times, and preferably 25 to 100 times, the final concentrations in the liquid composition of the present invention described above.

[0068] Deacylated gellan gum or its salt, and acidic polysaccharides (e.g., alginic acid) or their salts that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions may be sterilized, if necessary. Sterilization methods are not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, high-pressure steam sterilization (autoclave sterilization), and filter sterilization. The material of the filter used for 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), mixed cellulose esters, cellulose acetate, and polytetrafluoroethylene. 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 carried out when 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, are in a solid state or in a solution state.

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

[0070] The combination of high-pressure steam sterilization conditions is as follows: For example, 105 to 135°C, 0.12 to 0.32 MPa, and 1 to 60 minutes; Preferably, the heating temperature is 115°C to 130°C, the pressure is 0.17 to 0.27 MPa, and the time is 5 to 45 minutes; More preferably, the temperature is 118 to 123°C (eg, 121±1°C), the pressure is 0.19 to 0.23 MPa (eg, 0.21±0.1 MPa), and the time is 15 to 25 minutes (eg, 20±1 minutes).

[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, the deacylated gellan gum or a salt thereof, and the 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 in the liquid medium via metal cations (e.g., divalent metal cations such as calcium ions), thereby forming a three-dimensional network (irregular structure), and the liquid composition of the present invention can be obtained. Since a culture medium typically contains a sufficient concentration of metal cations (e.g., calcium ions) to allow 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 (irregular structure), 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 a liquid culture medium. Alternatively, the culture 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) may be added to the culture medium. Furthermore, the liquid composition of the present invention can also be prepared by mixing 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, with medium components (powdered medium or concentrated medium) in an aqueous solvent (e.g., water containing ion-exchanged water or ultrapure water).Examples of mixing methods include, but are not limited to, (1) mixing a liquid medium with a medium additive (solution), (2) adding deacylated gellan gum or a salt thereof and a solid (powder, etc.) 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, (3) mixing a powdered medium with a medium additive (solution), or (4) mixing a powdered medium with deacylated gellan gum or a salt thereof and a solid (powder, etc.) 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 with an aqueous solvent, etc. To prevent uneven distribution of the deacylated gellan gum or a salt thereof and the 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, mode (1) is preferred.

[0072] 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 crosslinked via divalent metal ions in a solvent (e.g., an aqueous solvent such as water or a liquid medium), or 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 crosslinked via divalent metal ions in a solvent, and a powdered medium in a solvent, the mixture may be heated to promote dissolution. The heating temperature may be, for example, 80°C to 130°C, preferably 100°C to 125°C (e.g., 121°C), which is sufficient for heat sterilization. After heating, the resulting 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 crosslinked via divalent metal ions is cooled to room temperature. By adding the above-mentioned metal cations (e.g., divalent metal cations such as calcium ions) to the solution (e.g., by adding the solution to a liquid medium), the deacylated gellan gum or its salt and the 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 will aggregate via the metal cations (e.g., divalent metal cations such as calcium ions), forming a three-dimensional network (irregular structure), and the liquid composition of the present invention can be obtained.Alternatively, when 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 are dissolved in a solvent (e.g., an aqueous solvent such as water or a liquid medium) containing the above-mentioned metal cation (e.g., a divalent metal cation such as calcium ion), the solution is heated (for example, to 80°C to 130°C, preferably 100°C to 125°C (e.g., 121°C)) and the resulting solution is cooled to room temperature, the deacylated gellan gum or a salt thereof and the 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 the metal cation (e.g., a divalent metal cation such as calcium ion), thereby forming a three-dimensional network (irregular structure).

[0073] Because deacylated gellan gum or a salt thereof has a relatively linear structural unit, multiple sugar chains bundle when added to a solvent (e.g., water), making it difficult to dissolve. However, 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 crosslinked via divalent metal ions is added, the deacylated gellan gum or a salt thereof is inhibited from bundling, resulting in relatively easy dissolution, due to the relatively bulky structure that contains both α1-4-linked L-glucuronic acid and β1-4-linked D-mannuronic acid uronic acids. Therefore, 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 can be dissolved in a solvent (e.g., water, an aqueous solvent such as a liquid medium) at relatively low temperatures (e.g., 0 to 37°C, preferably 10 to 30°C) without heating.

[0074] The method for producing the liquid composition of the present invention will be exemplified below, but is not limited thereto.

[0075] 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 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 maintains 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, and more preferably 10 to 30°C) until the mixture becomes transparent.

[0076] After dissolution, the solution is allowed to cool while stirring as necessary, and sterilized (for example, by autoclaving at 121°C for 20 minutes or by filtration). While stirring the medium (for example, using a homomixer), the sterilized aqueous solution is added to the medium and mixed uniformly with the medium. The method for mixing the aqueous solution with the medium is not particularly limited, and examples include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, or homogenizer.

[0077] To ensure that the deacylated gellan gum or its salt and the 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 are uniformly dispersed in the liquid medium, the liquid medium may be placed in a conical tube, stirred using a vortex mixer, 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 that 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 100-series Preparation Kit, it is possible to easily prepare the liquid composition of the present invention, in which a three-dimensional network (irregular 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, via metal cations (e.g., divalent metal cations such as calcium ions), is uniformly dispersed.

[0078] After mixing, the liquid composition of the present invention may be filtered using a filter having a pore size of 5 μm to 100 μm, preferably 5 μm to 70 μm, and more preferably 10 μm to 70 μm.

[0079] [Cell or tissue preservation method] The present invention also provides a method for preserving cells or tissues, which comprises preserving the cells or tissues in an unfrozen state in the liquid composition of the present invention. In a preferred embodiment, in the preservation method of the present invention, the cells or tissues can be preserved or transported in a suspended state (preferably in a suspended, stationary state) in the liquid composition of the present invention.

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

[0081] The morphology and state of cells and tissues to be preserved using the methods of the present invention can be freely selected by those skilled in the art. Specific examples of the state of cells to be preserved include, but are not limited to, a state in which cells are dispersed into single cells, a state in which cells are adhered to the surface of a carrier, a state in which cells are embedded inside a carrier, a state in which multiple cells have aggregated to form a cell cluster (e.g., a sphere), and a state in which two or more types of cells have aggregated to form a cell cluster (e.g., a sphere). Of these states, the state in which cell clusters (e.g., a sphere) have been formed is the most preferable state for preservation using the methods of the present invention, because cell-cell interactions and cell structures similar to those in vivo are reconstructed, cell functions can be maintained for a long period of time, and cell recovery is relatively easy. The cell mass is preferably a sphere containing mammalian stem cells (e.g., embryonic stem cells (ES cells), embryonic tumor 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, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, cancer stem cells) or progenitor cells. Examples of spheres in the present invention include aggregates formed from tens to hundreds of cells. Spheres can be produced by known methods.

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

[0083] In the preservation method of the present invention, desired cells or tissues are dispersed in the liquid composition of the present invention and then placed in a sealable container. Examples of such containers include, but are not limited to, flasks, plastic bags, Teflon (registered trademark) bags, tubes, and culture bags. To prevent leakage of the contents and contamination by bacteria and other external substances during preservation, the container containing the dispersion of cells or tissues 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 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, are assembled via metal cations (e.g., divalent metal cations such as calcium ions) to form a three-dimensional network (irregular 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, making it possible to store the cells or tissues in a floating, uniformly dispersed state (floating, static storage). On the other hand, this three-dimensional network is vulnerable to shear force due to the inclusion of an acidic polysaccharide (e.g., alginic acid) or salt thereof that maintains a random coil state in a divalent metal cation medium and is capable of crosslinking via divalent metal ions, and when a chelating agent is added as necessary to a preservation preparation containing the liquid composition of the present invention and cells or tissues, and shear force sufficient to disrupt this three-dimensional network is applied by pipetting or the like, the cell or tissue suspending property based on this three-dimensional network is quickly lost. Therefore, at the start of preservation, shear force such as pipetting or stirring is applied to disrupt the three-dimensional network (irregular structure) formed by the assembly of deacylated gellan gum or a salt thereof and an acidic polysaccharide (e.g., alginic acid) or salt thereof that maintains a random coil state in a divalent metal cation medium and is capable of crosslinking via divalent metal ions via metal cations (e.g., divalent metal cations such as calcium ions), thereby enabling cells or tissues to be quickly dispersed and suspended uniformly in the liquid composition of the present invention.Importantly, the destruction of the three-dimensional network (amorphous structure) by shear force (loss of the effect of maintaining the suspension of cells and tissues) is a reversible reaction, and if the resulting cell or tissue suspension is left standing, over time, the fragments of the destroyed three-dimensional network (amorphous structure) will reassemble through metal cations (e.g., divalent metal cations such as calcium ions), causing the three-dimensional network (amorphous structure) to regenerate and regain the ability to maintain the suspension of cells and tissues, allowing the cells and tissues to be preserved while maintaining their suspension.

[0085] The storage temperature is not particularly limited as long as the viability of the cells or tissues is maintained, but is typically 37°C or lower. A lower temperature can prevent a decrease in the viability of the cells or tissues during storage, but to prevent the cells or tissues from freezing, they are typically stored at a temperature above the melting point of the liquid composition of the present invention. Therefore, the storage temperature is typically maintained at 0 to 37°C, preferably 1 to 30°C, more preferably 15 to 30°C (e.g., room temperature storage at 15 to 25°C), even more preferably 22 to 28°C, and even more preferably 24 to 26°C (e.g., 25°C).

[0086] In order to enable preservation of cells or tissues in a suspended, static state, the temperature during storage is preferably a temperature at which the liquid composition of the present invention allows the cells or tissues to be preserved to be suspended and static.

[0087] The storage period is not particularly limited as long as the cells or tissues to be stored can be maintained in a viable state in the liquid composition of the present invention, but is usually 1 hour or longer (e.g., 12 hours or longer, 24 hours (1 day) or longer, 2 days or longer). The upper limit of the storage period is not particularly limited as long as the cells or tissues to be stored can be maintained in a viable state in the liquid composition of the present invention, but is usually 28 days or shorter (e.g., 21 days or shorter, 14 days or shorter, 7 days or shorter, 3 days or shorter). The storage period is set appropriately depending on the purpose of storage. During storage or transportation, it is preferable that the cells or tissues be maintained in a suspended, stationary state in the liquid composition of the present invention.

[0088] In one embodiment, cells or tissues are preserved in the liquid composition of the present invention in an environment accompanied by vibration. An example of an "environment accompanied by vibration" is the environment during transportation of the cells or tissues. That is, the preservation method of the present invention can also be understood as a method of transporting cells or tissues while preserving them in an unfrozen state in the liquid composition of the present invention.

[0089] The preservation method of the present invention allows cells and tissues to be maintained in a floating state, thereby avoiding damage to the cells and tissues due to detachment from the plate caused by vibration during transport or aggregation of cells or tissues that come into contact with each other due to sedimentation, and allows cells and tissues to be preserved in a state where their original functions are maintained.When spheres are preserved using the method of the present invention, the spheres can be maintained in a floating state, thereby avoiding aggregation of spheres due to vibration during transport and vibration, and allowing the spheres to be preserved while maintaining their morphology.

[0090] Retrieval of preserved cells or tissues The present invention also provides a method for efficiently recovering cells or tissues from a preservation preparation obtained by preserving the cells or tissues in the liquid composition of the present invention, which method comprises applying shear 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 crosslinked via divalent metal ions, are assembled via metal cations (e.g., divalent metal cations such as calcium ions) to form a three-dimensional network (irregular 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, making it possible to store the cells or tissues in a floating, uniformly dispersed state (floating, static storage). On the other hand, this three-dimensional network is vulnerable to shear force because it maintains a random coil state in a divalent metal cation medium and contains an acidic polysaccharide (e.g., alginic acid) or its salt that can be crosslinked via divalent metal ions. When a shear force sufficient to destroy this three-dimensional network is applied to a preservation preparation containing the liquid composition of the present invention and cells or tissues, with the addition of a chelating agent as necessary, the cell or tissue suspending property based on this three-dimensional network is quickly lost, and the cells or tissues become more likely to sediment by gravity. When the preservation preparation is centrifuged in this state, the cells or tissues contained therein easily sediment, and the cells or tissues can be recovered by removing the supernatant liquid composition.

[0092] The operation of applying shear force to the preserved preparation is not particularly limited, as long as it can destroy the three-dimensional network (irregular structure) formed by the aggregation of deacylated gellan gum or its salt, and acidic polysaccharides (e.g., alginic acid) or its salts that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions, via metal cations (e.g., divalent metal cations such as calcium ions), and examples of such operations include pipetting, filtration, stirring, and ultrasonic waves.

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

[0094] In addition, in order to allow the entire preserved preparation to be stirred quickly, it is preferable to inhale and exhale, for example, 1% or more of the volume of the preserved preparation, preferably 10% or more, more preferably 20% or more, even more preferably 30% or more, and even more preferably 50% or more in a single inhalation and exhalation.

[0095] In order to apply sufficient shear force to the preserved preparation, it is preferable to perform the inhalation and / or exhalation operation at a flow rate of, for example, 1 ml / sec or more, preferably 2 to 20 ml / sec, and more preferably 5 to 10 ml / sec.

[0096] The number of pipetting times is not particularly limited as long as it is sufficient to disrupt the three-dimensional network, but is usually at least one, preferably at least three, and more preferably at least five consecutive times. The more times pipetting is performed, the more reliably the three-dimensional network is disrupted, so this is preferable, and theoretically there is no upper limit, but if the number of pipetting times is too high, the viability of the cells or tissue decreases, so it is usually 50 times or less, preferably 20 times or less, and more preferably 15 times or less. The number of times pipetting is usually 1 to 50 times, preferably 3 to 20 times, and more preferably 5 to 15 times.

[0097] The size of the pores (pore diameter) of the filter is not particularly limited as long as it can destroy the three-dimensional network, but is usually 500 μm or less, preferably 200 μm or less, and more preferably 100 μm or less. The smaller the pore diameter, the stronger the shear force acting on the preserved preparation, and the more reliably the three-dimensional network can be destroyed. However, if the pore diameter is too small, the liquid composition will have difficulty passing through the filter. Therefore, the size of the pores (pore 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 large enough to allow cells or tissues in the preserved preparation to pass through. Here, "a size that allows cells or tissues to pass through" refers to a size that allows cells or tissues to pass through while maintaining their viability. For example, not only does the pore size of the filter be larger than the diameter of the cells or tissues to be preserved, but also includes situations in which cell clusters (e.g., spheres) or tissues in the preserved preparation pass through a filter with a pore size smaller than the diameter of the cells or tissues, thereby dividing the cells, cell clusters (e.g., spheres), or tissues into multiple cells, cell clusters (e.g., spheres), or tissues while maintaining their viability. Cell size cannot be generally defined because it depends on the cell type. However, typical cells with diameters of approximately 7.5 to 20 μm can easily pass through a filter with a pore size of 20 μm or more, preferably 40 μm, in the state of a single cell, while maintaining good viability. Therefore, from the perspective of efficiently disrupting the three-dimensional network while maintaining good cell or tissue viability, the pore size of the filter is preferably within the range of, for example, 20 to 200 μm, preferably 40 to 100 μm.

[0099] Filter materials include, but are not limited to, polyethylene, polypropylene, polyamide (nylon), polysulfone, polypropylene, acrylic, polylactic acid, cellulose mixed esters, polycarbonate, polyester, and glass. While properties such as polarity, electrostatic charge, and hydrophilicity vary depending on the material, there is little correlation between these properties and recovery rates, and a good recovery rate can be expected regardless of the material used. Polyamide (nylon), polyethylene, polyester, and glass are preferred due to their availability.

[0100] These filters may be commercially available products, and specific examples thereof include CellTrics filters (trademark) manufactured by Partec: pore sizes of 5 μm (product number 06-04-004-2323), 10 μm (product number 06-04-004-2324), 20 μm (product number 06-04-004-2325), 30 μm (product number 06-04-004-2326), 50 μm (product number 06-04-004-2327), 100 μm (product number 06-04-004-2328), and 150 μm (product number 06-04-004-2329); and Cell Strainer (trademark) manufactured by Becton Dickinson: pore sizes of 40 μm (product number 352340), 70 μm (product number 352350), and 100 μm (product number 352360). μm (model number 352360), AS ONE Corporation's Filcon S (trademark): 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).

[0101] The number of times the cells or tissues are passed through a filter may be one, but if necessary, the recovery rate of the cells or tissues can be improved by passing the cells or tissues through a filter multiple times. The number of times the cells or tissues are passed through a filter is usually 1 to 10 times.

[0102] When passing the cell or tissue preservation preparation through a filter multiple times, the procedure of passing the cell or tissue preservation preparation through a single filter and recovering the passed suspension may be repeated multiple times, or the cell or tissue preservation preparation may be passed through a multiple filter containing multiple (e.g., 3 to 5) stacked filter membranes. The use of a multiple filter is advantageous from the viewpoint of operational efficiency. When passing the cell or tissue preservation preparation through a 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 (e.g., 3 to 5) stacked filters with the same pore size (e.g., 40 to 100 μm) are used.

[0103] Stirring procedures include vortexing, inversion mixing, magnetic stirrer, paddles, etc. The vortexing speed is, for example, 200 to 3,000 rpm.

[0104] When shear force is applied to the preservation preparation, a chelating agent may be added to the culture preparation as needed. The addition of a chelating agent removes metal cations (preferably divalent metal cations such as calcium ions and magnesium ions) from the three-dimensional network contained in the liquid composition, loosening the bonds between polysaccharides (deacylated gellan gum or a salt thereof, and acidic polysaccharides (e.g., alginic acid) or a salt thereof that maintain a random coil state in a divalent metal cation medium and can be crosslinked via divalent metal ions) in the three-dimensional network via the metal cations, partially destroying the three-dimensional network and expected to improve the recovery rate of cells or tissues.

[0105] The chelating agent is not particularly limited as long as it is a compound capable of forming a complex with a divalent metal cation such as calcium ion or magnesium ion (preferably calcium ion), and examples thereof include citric acid or a salt thereof (e.g., trisodium citrate); EDTA or a salt thereof (e.g., edetate sodium salts such as EDTA2Na, EDTA3Na, and EDTA4Na); hydroxyethylethylenediaminetriacetate such as HEDTA3Na; EGTA or a salt thereof; pentetate (diethylenetriaminepentaacetate); phytic acid; phosphonic acids such as etidronic acid and salts thereof 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. From the viewpoint of improving the recovery rate of cells or tissues, citric acid or a salt thereof (e.g., trisodium citrate) or EDTA or a salt thereof (e.g., edetate sodium salts such as EDTA2Na, EDTA3Na, EDTA4Na) are preferred. Two or more types of chelating agents can also be used in combination. The combination of chelating agents is not particularly limited, but examples include a combination of citric acid or a salt thereof (e.g., trisodium citrate) and EDTA or a salt thereof (e.g., edetate sodium salts such as EDTA2Na, EDTA3Na, EDTA4Na).

[0106] The amount of chelating agent added 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 crosslinked via divalent metal ions) in the three-dimensional network contained in the liquid composition of the present invention, mediated by metal cations. For example, in the case of citric acid or a salt thereof (e.g., trisodium citrate), the final concentration immediately after addition is usually 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 a concern that it may affect the viability of cells or tissues, so the upper limit is usually 0.2 w / v% or less, more preferably 0.1 w / v% or less. In the case of EDTA or a salt thereof (e.g., edetate sodium salts such as EDTA2Na, EDTA3Na, and EDTA4Na), the final concentration immediately after addition is usually 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 a concern that it may affect the viability of cells or tissues. Therefore, the upper limit is usually 0.2 w / v% or less, more preferably 0.1 w / v% or less.

[0107] After adding the chelating agent to the preservation preparation, it is preferable to thoroughly stir the preservation preparation by applying shear force to the preservation preparation so that the chelating agent is uniformly distributed.

[0108] After the above pretreatment step, the resulting mixture containing cells or tissues is centrifuged to precipitate the cells or tissues, and fractions other than the cells or tissues (e.g., the supernatant liquid composition of the present invention) are removed, thereby finally recovering the cells or tissues from the cell or tissue preservation preparation. Techniques for precipitating cells or tissues by centrifugation are well known to those skilled in the art, and those skilled in the art can set appropriate conditions depending on the type of cell or tissue. Generally, centrifugation is performed at a centrifugal force of about 10 to 400 G to precipitate the cells or tissues and separate them from the supernatant.

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

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

[0111] [Test Example 1] Preparation of polysaccharide mixture One part by mass of sodium alginate (ALG) (Kimica Algin IL-2, manufactured by Kimica Co., Ltd.) and 99 parts by mass of purified water were added to a glass culture medium bottle, and the bottle was sterilized in an autoclave (121°C, 20 minutes) to prepare an ALG aqueous solution with a concentration of 1% by mass. Similarly, an aqueous solution of deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) with a concentration of 1% by mass was prepared. The ALG aqueous solution and the DAG aqueous solution were dispensed into conical tubes at a ratio of 2:1 (v / v), and the mixture was thoroughly mixed by pipetting using a disposable syringe equipped with a syringe needle to make it homogenous, thereby preparing a polysaccharide mixture.

[0112] [Test Example 2] Preparation of liquid composition Culture medium preparation kit (Nissan Chemical Industries FCeM (R)Liquid compositions of the present invention were prepared using a 50 mL (Sumitomo Bakelite) series Preparation Kit. Predetermined amounts of various culture media were dispensed into conical tubes (50 mL centrifuge tubes manufactured by Sumitomo Bakelite), and an adapter cap, a component of the kit, was attached. The tip of a disposable syringe filled with a predetermined amount of the polysaccharide mixture obtained in Test Example 1 was fitted into the cylindrical part of the adapter cap to connect it, and the syringe plunger was manually pressed, forcing the polysaccharide mixture in the syringe into a container and instantly mixing it with the culture medium to prepare a liquid composition of the present invention. The liquid compositions prepared are shown in Table 1.

[0113] [Table 1]

[0114] [Analysis] Viscosity measurement of liquid compositions 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 (Toki Sangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1°34´×R24) at 25°C and 100 rpm for 5 minutes. The average value of three measurements was 2.07 mPa s (1st measurement: 1.95 mPa s, 2.21 mPa s, 3rd measurement: 2.05 mPa s).

[0115] [Test Example 3] Cell preservation (cell type: NHDF) Normal human neonatal foreskin fibroblasts (NHDF, Kurabo Industries, Ltd.) were added to 152 × 10 4 Prepare a cell suspension containing 38 x 10 cells in four conical tubes. 4 After centrifugation (300 x g, 3 minutes) and removal of the supernatant, the cells were resuspended in 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 (Comparative Example 1 or Comparative Example 2) not containing DAG or ALG for comparison, and the cell suspension (20 x 10 4A total of 100 μL of the solution was dispensed into 18 1.5 mL round-bottom microtubes, capped, and stored at 25 (±1)°C. Three tubes were removed from each tube on the initial day of storage and on days 1, 4, 7, 14, and 21 of storage. The ATP content in the cells was quantified using CellTiter-Glo reagent (Promega) on a plate reader (infinite M200 PRO, Tecan). The RLU value measured on the initial day of storage was used as the reference (100% cell viability), and the RLU values ​​measured after each day of storage were compared to calculate the cell viability over time to evaluate cell preservation. The average values ​​measured three times in the above test are shown in Table 2.

[0116] [Table 2]

[0117] As can be seen from Table 2, in Comparative Example 1, which did not contain a polysaccharide composition, the survival rate decreased to 30%, whereas in DHb086, a liquid composition containing DAG and ALG, approximately 70% survival was confirmed even after 3 weeks, and in DHb090, 50% survival was confirmed after 4 days. This confirms that the liquid composition containing DAG and ALG exhibits a cell preservation effect.

[0118] [Test Example 4] Cell preservation (cell type: NHDF) Normal human neonatal foreskin fibroblasts (NHDF, Kurabo Industries, Ltd.) were added to 310 × 10 4 Prepare a cell suspension containing 62 x 10 cells in five conical tubes. 4 After centrifugation (300 x g, 3 minutes) and removal of the supernatant, the cells were resuspended in 3.1 mL of a liquid composition containing DAG and ALG (total concentration 0.010, 0.013, 0.016, 0.020 (w / v)%) shown in Table 1 (DHb084, DHb085, DHb086, or DHb087 in Table 1) or a liquid composition not containing DAG or ALG (Comparative Example 1) for comparison, and the cells were resuspended in 3.1 mL of a cell suspension (20 x 10 4A total of 100 μL of the solution was dispensed into five wells of a 96-well U-bottom cell culture plate (Sumitomo Bakelite Co., Ltd., MS-309UR), and six plates were placed in each well at 25 (±1)°C. The ATP content in each of the five wells was quantified using CellTiter-Glo reagent (Promega) and a plate reader (infinite M200 PRO, Tecan Biosciences) on the initial day of storage and on days 1, 3, 7, 14, and 21 of storage. The RLU values ​​measured on the initial day of storage were used as the reference (100% cell viability) to compare with the RLU values ​​measured after each day of storage. Cell viability was calculated over time to evaluate cell preservation. The average values ​​for the five measurements are shown in Table 3.

[0119] [Table 3]

[0120] As can be seen from Table 3, at any polysaccharide concentration, the survival rate was approximately 70% or more after 3 weeks, confirming the high cell preservation effect of the liquid composition containing DAG and ALG.

[0121] [Test Example 5] Cell preservation (cell type: h-MSC) Human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) were added to 104 × 10 4 Prepare a cell suspension containing 26 x 10 cells in four conical tubes. 4 After centrifugation (300 x g, 3 minutes) and removal of the supernatant, the cells were resuspended in 1.3 mL of a liquid composition containing DAG and ALG (total concentration 0.016 (w / v)%) shown in Table 1 (Examples DHb086 to DHb090) or a liquid composition not containing DAG or ALG (Comparative Examples 1 and 2) for comparison, and the cells were resuspended in a cell suspension (20 x 10 4A 100 μL aliquot of the solution was placed into twelve 1.5 mL round-bottom microtubes, capped, and stored at 25 (±1)°C. Three tubes were removed from each tube on the first day of storage, the third day, and the seventh day after storage, and the amount of ATP in the cells was quantified using CellTiter-Glo reagent (Promega) on a plate reader (infinite M200 PRO, Tecan). The RLU value measured on the first day of storage was used as the reference (100% cell viability), and the RLU values ​​measured after each day of storage were compared to calculate the cell viability over time to evaluate cell preservation. The average values ​​of the three measurements taken in the above test are shown in Table 4.

[0122] [Table 4]

[0123] As shown in Table 4, approximately 70% of DHb086 and approximately 60% of DHb090 cells remained viable even after one week, and the liquid composition containing DAG and ALG showed an approximately two-fold improvement in viability, confirming the excellent cell preservation effect of the liquid composition containing DAG and ALG.

[0124] [Test Example 6] Cell preservation test with varying cell seeding density and storage temperature (cell type: h-MSC) Human bone marrow-derived mesenchymal stem cells (h-MSC, manufactured by LONZA) were added to 510 × 10 4 Prepare 17 mL of cell suspension containing 3 x 10 cells and place in four conical tubes. 4 Cells (0.1mL), 30 x 10 4 Cells (1 mL), 150 x 10 4 Cells (5 mL), 300 x 10 4 The cells (10 mL) were dispensed into each well. After centrifugation (300 x g, 3 minutes) and removal of the supernatant, the cells were resuspended in a liquid composition (DHb086) containing DAG and ALG (total concentration 0.016 (w / v)%) shown in Table 1, and the resulting cell suspensions were prepared at various cell densities (1 x 10 4 cells / mL, 10×10 4 cells / mL, 50×10 4 cells / mL, 100×104 A total of 100 μL of the solution was dispensed into 24 1.5 mL round-bottom microtubes, capped, and stored at 25 (±1)°C or 37°C. Three tubes were removed from each tube on the first day of storage and on days 1, 3, and 7 after storage. The ATP content in the cells was quantified using CellTiter-Glo reagent (Promega) on a plate reader (infinite M200 PRO, Tecan). The RLU value measured on the first day of storage was used as the reference (100% cell viability), and the RLU values ​​measured after each day of storage were compared to calculate the cell viability over time to evaluate cell preservation. The average values ​​of the three measurements taken in the above test are shown in Table 5.

[0125] [Table 5]

[0126] As can be seen from Table 5, the liquid composition containing DAG and ALG exhibited excellent cell preservation properties at all cell seeding densities in the storage test at 25°C. However, the survival rate significantly decreased in the storage test at 37°C, 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 added to 32.4 × 10 4 A cell suspension containing 32.4 × 10 cells was prepared and placed in a 6-well plate for sphere preparation (Elplasia Spheroid Generators MPc500, Kuraray Co., Ltd.). 4h-MSC spheres were prepared by adding 4 mL of cell suspension per well and culturing for 3 days in serum-supplemented DMEM-Low Glucose medium at 37°C and 5% CO2. Six hundred fifty 500 μm diameter pinholes were drilled into the bottom of the wells, allowing cells to be seeded at a density of 500 cells per hanging drop-shaped pinhole, resulting in approximately 650 spheres per pinhole. The resulting spheres were collected in a 15 mL conical tube, centrifuged (100 x g, 1 minute), and the supernatant was removed. Afterwards, 1.3 mL of a liquid composition (DHb087) containing DAG and ALG (total concentration 0.020 (w / v)%), as shown in Table 1, was added and resuspended to prepare a sphere suspension (50 cells / 100 μL). This suspension was dispensed into twelve 1.5 mL round-bottom microtubes (100 μL each), capped, and stored at 25 (±1)°C. Three tubes were removed from each cell on the first day of storage and on days 3, 5, and 7 after storage. The amount of ATP contained in the cells was quantified using CellTiter-Glo reagent (Promega) on a plate reader (infinite M200 PRO, Tecan). The RLU value measured on the first day of storage was used as the reference (100% cell viability), and the RLU values ​​measured after each day of storage were compared. The cell viability over time was calculated to evaluate cell preservation. The average values ​​of the three measurements in the above test are shown in Table 6. Figure 1 shows the appearance of the sphere-preserved suspension after 7 days of storage, as well as photographs of the spheres before and after storage.

[0128] [Table 6]

[0129] As can be seen from Table 6, the spheres survived for 5 days without dying, and 80% or more survived for 7 days. When the spheres were stored in the medium of Comparative Example 1, the spheres aggregated together and necrosis occurred inside the aggregates, so the medium was excluded from the test (data not shown).

[0130] As can be seen from FIG. 1, the sphere shape was maintained before and after storage, and it was confirmed that the spheres remained floating in the liquid composition containing DAG and ALG even after 7 days of storage.

[0131] [Test Example 8] Sphere preservation in a vibration environment (cell type: h-MSC) Human bone marrow-derived mesenchymal stem cells (h-MSC, LONZA) were added to 64.8 × 10 4 A cell suspension containing 32.4 × 10 cells was prepared and placed in a 6-well plate for sphere preparation (Elplasia Spheroid Generators MPc500, Kuraray Co., Ltd.). 4 h-MSC spheres were prepared by adding 4 mL of the cell suspension to two wells and culturing them for 3 days in serum-supplemented DMEM-Low Glucose medium at 37°C and 5% CO2. Six hundred fifty 500 μm diameter pinholes were drilled into the bottom of the wells, allowing cells to be seeded at a density of 500 cells per pinhole, resulting in approximately 650 spheres per well. The resulting spheres were collected in 15 mL conical tubes and centrifuged (100 x g, 1 minute) to remove the supernatant. After resuspending the spheres in 1.3 mL of a liquid composition containing DAG and ALG (total concentration 0.020 (w / v)%) (DHb087) or a comparative liquid composition without DAG or ALG (Comparative Example 1), as shown in Table 1, to prepare sphere suspensions (50 spheres / 100 μL). Each tube was dispensed in 100 μL aliquots into twelve 1.5 mL round-bottom microtubes, capped, and stored at 25 (±1) °C in a shaking environment (high-speed shaker ASCM-1 (equipped with a 1.5 mL tube rack, 300 rpm), AS ONE Corporation). Three tubes were removed from each tube on the first day of storage, the third day, and the seventh day after storage, and the ATP content in the cells was quantified using CellTiter-Glo reagent (Promega) on a plate reader (infinite M200 PRO, Tecan Biosciences). The RLU value measured on the first day of storage was used as the reference (100% cell viability), and the RLU values ​​measured after each day of storage were compared to calculate the cell viability over time to evaluate cell preservation. The average values ​​from the three measurements above are shown in Table 7.

[0132] [Table 7]

[0133] A preservation test of h-MSC spheres was conducted under vibration conditions simulating the environment during transportation, and the liquid composition containing DAG and ALG showed good cell preservation properties even under vibration conditions, as shown in Table 7. In Comparative Example 1, the aggregation of spheres was somewhat suppressed due to vibration, but a decrease in viability was observed.

[0134] [Reference Example 1] Preparation of polysaccharide mixture A glass culture medium bottle was filled with 1 or 2 parts by weight of sodium alginate (ALG) (Kimica Algin, manufactured by Kimica Co., Ltd.) and 99 or 98 parts by weight of purified water, and the bottle was sterilized in an autoclave (121°C, 20 minutes) to prepare an aqueous solution of ALG with a concentration of 1% or 2% by weight. Similarly, aqueous solutions of deacylated gellan gum (DAG) (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) with concentrations of 1% by mass and 2% by mass were prepared. A predetermined amount of the ALG aqueous solution and the DAG aqueous solution were dispensed into a microtube, and the mixture was thoroughly mixed by pipetting using a disposable syringe equipped with a syringe needle to make it homogenous, thereby preparing a polysaccharide mixture.

[0135] [Reference Example 2] Preparation of liquid composition (1) Preparation of liquid composition using a vortex mixer A predetermined amount of medium was dispensed into a conical tube (Sumitomo Bakelite 15 mL, 50 mL, or 225 mL centrifuge tube) and stirred in an open state using a vortex mixer. A predetermined amount of the polysaccharide mixture obtained in Reference Example 1 was filled in the tube and vigorously added to the medium using a disposable syringe (Terumo Terumo Syringe) equipped with a syringe needle (Fuchigami Kikai FN5200), to prepare a liquid composition.

[0136] (2) Culture medium preparation kit (Nissan Chemical Industries FCeMTM Preparation of liquid compositions using the -series Preparation Kit A predetermined amount of medium was dispensed into a conical tube (Sumitomo Bakelite 50 mL centrifuge tube), and an adapter cap, a component of the kit, was attached. The tip of a disposable syringe filled with a predetermined amount of the polysaccharide mixture obtained in Reference Example 1 was fitted into the cylindrical part of the adapter cap to connect it, and the syringe plunger was manually pressed to forcefully eject the polysaccharide mixture from the syringe into the container and bring it into contact with the medium, thereby preparing a liquid composition.

[0137] [Reference Example 3] Confirmation of floating action Polystyrene beads (diameter 500-600 μm, manufactured by Polysciences Inc.) for simulating floating cells were added to the liquid composition prepared in Reference Example 2 and stirred. Ten minutes after stirring was stopped, the dispersion state of the beads in the liquid was visually confirmed. 2+ When a sufficient amount of the structure formed by crosslinking via crosslinking via cations (e.g., cations) is properly and finely dispersed in the liquid, the beads will also be dispersed and remain suspended in the liquid. On the other hand, if the structure is not sufficiently dispersed, the beads will also settle accordingly. The state of dispersion of the beads is represented by ○ when they are well dispersed and suspended, △ when they are dispersed but some have settled, and × when all the beads have settled.

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

[0139] [Table 8]

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

[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 cultured at 1800 × 10 4 Cell preparation: 300 x 10 4 After centrifugation (300 x g, 3 minutes) and removal of the supernatant, 30 mL of liquid compositions (Examples C369 to C373 in Table 10) containing DAG and ALG (total concentration 0.015 (w / v)%) at various ratios were added and gently stirred to prepare cell suspensions (10 x 10 4 10 × 10 cells / mL) were placed in a 24-well cell culture plate (Sumitomo Bakelite Co., Ltd.). 4 One mL of cell suspension was added per well and cultured at 37°C under 5% CO2 for one week. After incubation, the cell concentration of the cell suspension was measured using a cell counter (TC-20, BIO-RAD). The suspension was transferred to a 1.5 mL microtube and homogenized by pipetting 20 times using a micropipette (Thermo Scientific, ClipTip 1000 μL) set to 0.2 mL. The cells were then centrifuged (300 x g, 3 minutes), 1.1 mL of the supernatant was removed, and 0.9 mL of DMEM-LG containing 10% fetal bovine serum was added for resuspension. The ATP content in the cells was quantified using CellTiter-Glo (Promega) on a plate reader (Tecan). The RLU value obtained by measuring the cell suspension after incubation before cell harvesting was used as the reference (100% cell recovery) and compared with the RLU value obtained after cell harvesting with the addition of suspension inhibitor. All of the above tests were carried out three times, and the average values ​​are shown in the table.

[0143] [Table 10]

[0144] [Reference Example 5] Examination of the number of pipetting Normal human neonatal foreskin fibroblasts (NHDF, Kurabo Industries) in the logarithmic growth phase were cultured at 1440 × 10 4Cells were prepared and suspended in 48 mL of the liquid composition of Example C371 in Table 10, and 30 x 10 4 Cells (1 mL) were dispensed into 24-well cell culture plates (Sumitomo Bakelite Co., Ltd.) and cultured at 37°C under 5% CO2 for 3 days. The cell concentration of the cultured cell suspension was measured using a cell counter (TC-20, BIO-RAD). The suspension was then transferred to a 1.5 mL microtube and pipetted repeatedly using a micropipette (Thermo Scientific, ClipTip 1000 μL) set to 0.2 mL. The cells were then centrifuged (300 x g, 3 minutes). 1.1 mL of the supernatant was removed and resuspended in 0.9 mL of DMEM-LG containing 10% fetal bovine serum. The ATP content in the cells was quantified using CellTiter-Glo (Promega) on a plate reader (Tecan) to calculate the cell recovery rate. Each test was performed five times, and the average values ​​are shown in the table.

[0145] [Table 11]

[0146] [Reference Example 6] Addition of a chelating agent Normal human neonatal foreskin fibroblasts (NHDF, Kurabo Industries) in the logarithmic growth phase were cultured at 450 × 10 4 The cells were prepared and suspended in 15 mL of the liquid composition of Example C371 in Table 10, and 30 x 10 4Cells (1 mL) were dispensed into 24-well cell culture plates (Sumitomo Bakelite Co., Ltd.) and cultured for 3 days at 37°C under 5% carbon dioxide. After measuring the cell concentration of the cultured cell suspension 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 (a mixed aqueous solution of 0.033 (w / v)% EDTA-2Na and 0.007 (w / v)% sodium citrate) was added. The mixture was pipetted 0 or 10 times using a micropipette (Thermo Scientific, ClipTip 1000 μL) with a 0.2 mL aspiration and dispensing volume. The cells were then centrifuged (300 x g, 3 minutes), 1.1 mL of the supernatant was removed, and 0.9 mL of 10% fetal bovine serum-containing DMEM-LG was added to resuspend the cells. The amount of ATP contained in the cells was quantified using CellTiter-Glo (Promega) on a plate reader (Tecan) to calculate the cell recovery rate. All of the above tests were performed three times, and the average values ​​are shown in the table.

[0147] [Table 12]

[0148] [Test Example 7] Jurkat cell proliferation Human T-cell leukemia-derived cells (Jurkat E6.1, DS Pharma Biomedical Co., Ltd.) in the logarithmic growth phase were cultured at 240 × 10 4 Prepare 40 x 10 cells 4 Each cell was centrifuged (300 x g, 3 minutes) and the supernatant was removed. Then, 8 mL of 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 but not ALG (Comparative Example DHb024 in Table 13) were added and gently stirred to prepare a cell suspension (5 x 10 4 0.5 × 10 cells / mL) were placed in a 96-well U-bottom cell culture plate (Sumitomo Bakelite Co., Ltd., MS-309UR). 40.1 mL of cell suspension was added per well, and the cells were cultured at 37°C under 5% carbon dioxide for 1 or 4 days. The cell counts before and after culture were compared using a plate reader (Tecan, infiniteM200PRO) with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) to measure the amount of ATP contained in the cells. All of the above tests were performed four times, and the average values ​​are shown in the table.

[0149] [Table 13]

[0150] As a result of the evaluation, the liquid composition of the present invention achieved cell proliferation comparable to that of the comparative example.

[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 cultured at 86.4 × 10 4 The cells were prepared and centrifuged (300 x g, 3 minutes) to remove the supernatant. Then, 8 mL of a liquid composition containing DAG and ALG (mass ratio 1:0.5) at various concentrations (Examples E041 and E045, E047, and E048 in Table 14) and a liquid composition containing DAG but not ALG (Comparative Example E049 in Table 14) were added and gently stirred to prepare a cell suspension (5 x 10 4 0.5 × 10 cells / mL) were placed in a 96-well U-bottom cell culture plate (Sumitomo Bakelite Co., Ltd., MS-309UR). 4 0.1 mL of cell suspension was added per well, and the cells were cultured at 37°C under 5% carbon dioxide for 1 or 4 days. The cell counts before and after culture were compared using a plate reader (Tecan, infiniteM200PRO) with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) to measure the amount of ATP contained in the cells. All of the above tests were performed six times, and the average values ​​are shown in the table.

[0152] [Table 14]

[0153] As a result of the evaluation, the liquid composition of the present invention achieved cell proliferation comparable to that of the comparative example.

[0154] [Reference Example 9] Cell recovery at a 10 mL scale Liquid composition of Example E041 in Table 14 10 x 10 4 A549 cells were seeded at 1000kJ / mL and cultured at 37°C under 5% CO2 for two days. After culture, 10 mL aliquots of the cell suspension were added to 1 mL of a chelating agent (a mixture of 0.033 (w / v)% EDTA-2Na and 0.007 (w / v)% sodium citrate) and immediately passed through cell strainers (40 μm, 70 μm, 100 μm, Falcon® cell strainers) and centrifuged (3 min) at various conditions (50 × g, 100 × g, 300 × g, g = gravitational acceleration). Cell counts before and after the cell harvesting procedure were compared using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) on a plate reader (Tecan, infinite M200PRO) to measure the ATP content in the cells.

[0155] [Table 15]

[0156] The evaluation results showed 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. Storing spheres in the liquid composition of the present invention prevents aggregation of the spheres and suppresses the occurrence of necrosis within the spheres.

[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.

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

Claims

1. comprising deacylated gellan gum or a salt thereof, alginic acid or a salt thereof, and a metal cation; the concentration of the deacylated gellan gum or a salt thereof in the liquid composition is 0.002 to 0.01 (w / v)% in terms of free form deacylated gellan gum, the concentration of the alginic acid or a salt thereof is 0.004 to 0.1 (w / v)% in terms of free form, and the mass ratio of the alginic acid or a salt thereof to the deacylated gellan gum or a salt thereof is 1 or more in terms of free form; A liquid composition for preserving cells or tissues in a non-frozen state.

2. 2. The liquid composition of claim 1, wherein the metal cation is a calcium ion.

3. 3. The liquid composition according to claim 1, wherein the alginic acid or a salt thereof has been subjected to autoclaving.

4. The liquid composition according to any one of claims 1 to 3, comprising a three-dimensional network formed by the assembly of deacylated gellan gum or a salt thereof and alginic acid or a salt thereof via a metal cation.

5. A method for preserving cells or tissues, comprising preserving the cells or tissues in a non-frozen state in the liquid composition according to any one of claims 1 to 4.

6. 6. The method of claim 5, wherein the cells or tissue are preserved in a suspended state in the liquid composition.

7. The method according to claim 5 or 6, wherein the cells or tissues are stored at 1°C to 30°C.

8. The method according to any one of claims 5 to 7, wherein the cells or tissues are stored in a sealed container.

9. The method according to any one of claims 5 to 8, wherein the cells or tissues are preserved in an environment accompanied by vibration.

10. The method of any one of claims 5 to 9, wherein the cells are preserved in the form of spheres.

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