Zwitterion-containing composition, and use thereof

Abstract

The present invention addresses the problem of providing a composition with which it becomes possible to improve the recovery rate of spheroids after storage by minimizing the damage to the spheroids under non-cryopreservation.　The present invention relates to a composition for non-cryopreservation of a multicell cluster, the composition containing one or more types of aprotic zwitterionic compounds (A) and two or more types of additives (X) that are compatible with the components (A).

Description

Zwitterion-containing compositions and uses thereof

[0001] The present invention relates to a composition used for non-cryopreserving cultured cell systems of multicellular populations such as spheroids, organoids, and tissues, and uses thereof.

[0002] Regenerative medicine is expected to be a promising treatment for intractable diseases such as myocardial infarction, stroke, and liver cirrhosis. One approach to regenerative medicine is to transplant cells into the affected area to be regenerated. However, transplanting spheroids rather than single cells is expected to increase the rate of engraftment due to the reduced risk of apoptosis. Furthermore, it has been reported that spheroids containing coexisting α and β cells from pancreatic islets of Langerhans significantly improved insulin production. Organoids are expected to have functions more similar to those of organs than spheroids, and thus are attracting increasing attention for their use in regenerative medicine. In addition to regenerative medicine, the use of spheroids and organoids in drug discovery is also being considered due to the increasing restrictions on animal testing. Furthermore, attempts are being made to extract cell populations from patient sites to select effective therapeutic agents, such as anticancer drugs. Currently, regenerative medicine is performed by autologous transplantation of spheroids. In other words, current regenerative medicine involves generating iPS cells from the patient's cells in the hospital, then preparing spheroids and transplanting them. Therefore, this procedure is only available at select large hospitals equipped with state-of-the-art cell culture facilities. Furthermore, the process can take several months and is costly. Despite the promise of spheroid transplantation, it has not yet reached widespread use. While autologous transplantation is considered a safe method considering factors such as immune responses, it is costly and not necessarily a commercially viable technique. To further advance regenerative medicine, allogeneic spheroid transplantation is an option. However, this requires industrial production of spheroids. This would allow spheroids to be manufactured at facilities around the world and delivered to hospitals as needed. This would shorten the time required for spheroid therapy, significantly reducing the cost of regenerative medicine.

[0003] One of the challenges in achieving this is preserving spheroids and the like for a certain period of time. One method for preserving spheroids is cryopreservation, which allows cells and microorganisms to be preserved for long periods of time. One known invention involves cryopreservation of spheroids at −196°C using a cryopreservative such as dimethyl sulfoxide (DMSO) and an additive such as vitamin E (Patent Document 1). DMSO is classified as Class 3 by the United States Food and Drug Administration (FDA) and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), which is equivalent to ethanol and other organic solvents, and is considered to have little or no significant toxicity. However, it is known to be relatively harmful as an organic solvent (Non-Patent Document 1). Therefore, the use of amino acid-based surfactants has been proposed as a way to reduce the amount of DMSO used (Patent Document 2). The present inventors have already proposed a nonionic zwitterionic compound for cryopreservation (Patent Document 3).

[0004] WO 98 / 35021 JP 2023-112417 A WO 2020 / 230721

[0005] Tuncer, S. et al. Low dose dimethyl sulfoxide driven gross molecular changes have the potential to interfere with various cellular processes. Sci. Rep. 8, 14828 (2018).

[0006] To realize regenerative medicine in which spheroids are produced at spheroid production centers around the world and custom-made spheroids are provided to local medical centers, non-cryopreservation of spheroids at a general-purpose freezing temperature of -25°C is more effective than cryopreservation, not only because it causes less damage to the spheroids themselves but also because it is more useful in terms of the widespread use of social infrastructure. The objective of the present invention is to enable storage for one month, minimize damage to spheroids during non-cryopreservation, and achieve a recovery rate of viable cells of 90% or more after storage. No cryopreservation composition capable of forming a non-cryopreservation medium that can be used for such purposes is known.

[0007] The present inventors conceived the idea that a composition that does not freeze even at -25°C or below, does not have a melting point, does not vitrify, and does not supercool at storage temperatures, could be a candidate for solving the above-mentioned problems. They discovered that by using an aprotic zwitterionic compound in combination with two or more specific additives, a composition for non-cryopreservation of multicellular populations can be obtained that does not have a melting point, does not vitrify, does not supercool, and does not freeze even at -25°C or below, and thus completed the present invention.

[0008] The present invention provides the following inventions [1] to [4]: ​​[1] A composition for non-cryopreservation of a multicellular population, comprising one or more aprotic zwitterionic compounds (A) and two or more additives (X) compatible with component (A). [2] The aprotic compound (A) is a compound represented by the general formula (1):

[0009]

[0010] (R 1 represents a linear alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, or a linear C alkyl group -(OCH2CH2)m-; A represents a cation portion of a zwitterion, and represents a cation selected from an imidazolium cation, a phosphonium cation, an ammonium cation, a sulfonium cation, a pyrazolium cation, a pyridinium cation, a pyrrolidinium cation, a morpholinium cation, a cyclopropenium cation, and a piperidinium cation; R2 represents an alkylene group having 1 to 4 carbon atoms; R 3 represents an alkylene group having 2 to 4 carbon atoms; m represents a number of 1 or 2; n represents a number of 0 to 10; B represents an anion selected from -SO3-, -COO-, -OP=O(H)O-, -OP=O(CH3)O-, -OP=O(OR4)O-, and -OP=O(OH)O-; and R4 represents an alkyl group having 1 to 8 carbon atoms which may have a heteroatom. [3] The non-cryopreservable composition according to [1] or [2], wherein the additive X is an additive selected from the group consisting of: (i) a small molecule compound having no hydroxyl group or a planar compound; (ii) a compound having one or more hydroxyl groups; (iii) a peptide or a protein; (iv) a polymer compound; (v) a surfactant; and (vi) a zwitterionic polymer or zwitterionic oligomer represented by the following general formula (2):

[0011]

[0012] (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, and are —COO—, —SO—, —OP═O(H)O—, —OP═O(CH)O—, and —OP═O(OR 5 ) O- (wherein R 5represents a hydrogen atom or a methyl group); Z represents a hydrogen atom, an aromatic hydrocarbon group of 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a straight-chain or branched alkyl group of 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m, and n each represent a number indicating the content ratio of each repeating unit and satisfying the conditions 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s, and t each represent an integer of 0 to 6. [4] A method for preserving a multicellular population under non-freezing conditions, using the composition according to any one of [1] to [3].

[0013] By using the non-cryopreservation composition of the present invention, the multicellular population can be preserved in an unfrozen state because it does not have a melting point at storage temperatures, does not vitrify, does not supercool, and does not freeze even at temperatures below −25° C. Therefore, regenerative medicine using the multicellular population becomes industrially feasible.

[0014] 1 shows DSC charts of compositions prepared by varying the OE2imC3C / DMSO / EG ratios: 10 / 5 / 5, 10 / 10 / 10, 10 / 15 / 15, 10 / 20 / 20, and 10 / 25 / 25. The charts show the recovery rate of viable cells from MAF1 spheroids stored in OE2imC3C / DMSO / EG at −25° C. for 3 days, and the viability of the viable cells after storage relative to the cell population including dead cells. This figure shows the recovery rate of viable cells when MAF1 spheroids were stored at −25° C. for 20 days using the compositions OE2imC3C / DMSO / EG (10 / 20 / 20), C1imC3C / DMSO / EG (10 / 20 / 20), and C4imC3S (20 / 20 / 20), and the viability of viable cells after storage relative to the cell population including dead cells. Regarding the recovery rate after storing MAF1 in a composition consisting of OE2imC3C / DMSO at −25° C. for 3 days, the figure shows that the recovery rate was poor when the composition was solid (S) at the storage temperature, and high when the composition was liquid (L). 1 shows the recovery rate of MAF1 spheroids after storing 10 / 10, 10 / 20, 10 / 30, 10 / 40, and 10 / 50 compositions consisting of OE2imC3C / DMSO for 3 days at −25° C. This figure shows the time course of recovery of viable cells when MAF1 spheroids were stored for 30 days at −25° C. using an OE2imC3C / DMSO / EG (10 / 20 / 20) composition. This figure shows the time course of recovery of viable cells when 5555 spheroids were stored for 30 days at −25° C. using an OE2imC3C / DMSO / EG (10 / 20 / 20) composition. This figure shows the time course of recovery of viable cells when BOSC spheroids were stored for 30 days at −25° C. using an OE2imC3C / DMSO / EG (10 / 20 / 20) composition. 1 shows the time course of viable cell recovery when MAF1 cells were stored for 30 days at −25° C. using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20). 2 shows the time course of viable cell recovery when 5555 cells were stored for 30 days at −25° C. using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20).This figure shows the change in viable cell recovery rate when BOSC cells were stored at −25°C for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20). This figure shows the change in viable cell recovery rate when MAF1 spheroids were stored at −25°C for 30 days using a composition that uses 0.9% NaCl instead of the liquid zwitterion. This figure shows an optical microscope photograph (right) of MDA-MB-231 cells stored in a 10% by mass OE2imC3C aqueous solution at −80°C, and an optical microscope photograph (left) of MAF1 cell spheroids stored in medium at room temperature. This figure shows the difference in cell recovery rate when MAF1 spheroids were stored at −25°C for 10 days using test solutions containing different compositions of OE2imC3C, OE2imC3S, C1imC3C, and C1imC3S in addition to DMSO (20 wt%) and EG (20 wt%). This figure shows the difference in cell recovery rate when MAF1 spheroids were treated for 1 hour at 0°C with test solutions containing different compositions of OE2imC3C, OE2imC3S, C1imC3C, and C1imC3S in addition to DMSO (20 wt%) and EG (20 wt%).

[0015] In the present invention, "multicellular population" refers to a collection of cells such as spheroids, organoids, cell sheets, tissue structures, organ slices, and organs. In the present invention, "spheroid" refers to a three-dimensional structure of one or more types of cells. The spheroids in the present invention are spherical and have a diameter of 0.01 to 0.1 mm. Spheroids are formed from iPS cells and then differentiated, or iPS cells are differentiated and then formed into spheroids, and the resulting spheroids are transplanted into patients. Spheroids can be obtained by three-dimensional culture of single cells or multiple cells together. Extracellular matrix formation, not observed in single-cell cultures, is observed between cells, and cell-cell interactions are sometimes observed, making the spheroids more similar to in vivo tissues than two-dimensional cell cultures. These matrices serve as a scaffold for cells to move within the spheroid, just as cells move within biological tissues. Therefore, spheroids can also be used as an improved culture model for cell migration, differentiation, survival, and proliferation. Furthermore, in two-dimensional culture, cells are only partially polarized, but in three-dimensional cell culture, cell polarization may be observed throughout the entire cell. It is also said that cells grown in three dimensions exhibit different gene expression than cells grown in two dimensions. In the present invention, "organoid" refers to a three-dimensional structure of cells that, if it is an organ, exhibits a structure more similar to that of an organ. While only a relatively simple extracellular matrix exists in spheroids, more complex intercellular interactions are observed in organoids via the extracellular matrix. In the present invention, "tissue structure" refers to a structure composed of cells and tissues. This includes sections of organs such as kidneys, livers, and testes, and may also serve as materials for forming organoids.

[0016] The present invention provides a non-cryopreservation composition that can be used for non-cryopreservation without the need for cryopreservation at low temperatures, even in areas where the spheroid formation site and the use site are far apart or where the social infrastructure lacks advanced cryopreservation facilities. In the present invention, "storage under non-frozen conditions" ("non-cryopreservation") refers to storage in a state where neither the water in the culture medium surrounding the cell population nor the water inside the cell population is frozen. Non-cryopreservation is performed in a storage space at liquid nitrogen temperatures to 37°C, preferably -85°C to room temperature, and more preferably -50°C to 0°C. Considering the use of food cold chains, it is preferable for the composition to be non-frozen at temperatures between -85°C and 4°C, preferably -30°C to -4°C, and more preferably -30°C to -15°C. Even if the composition is unfrozen in a stationary state, ice crystals may precipitate due to vibration during transportation. Therefore, the composition used in the present invention must not be supercooled at storage temperatures.

[0017] One aspect of the present invention is a composition for non-cryopreservation of a multicellular population, which comprises one or more aprotic zwitterionic compounds (A) and two or more additives (X) compatible with component (A). Each component is described below.

[0018] (Aprotic Zwitterionic Compound (A)) The aprotic zwitterionic compound (A) used in the present invention is a compound represented by the general formula (1):

[0019]

[0020] (R 1 represents a linear alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, or a linear C alkyl group -(OCH2CH2)m-; A represents a cation portion of a zwitterion, and represents a cation selected from an imidazolium cation, a phosphonium cation, an ammonium cation, a sulfonium cation, a pyrazolium cation, a pyridinium cation, a pyrrolidinium cation, a morpholinium cation, a cyclopropenium cation, and a piperidinium cation; R 2 represents an alkylene group having 1 to 4 carbon atoms; R 3represents an alkylene group having 2 to 4 carbon atoms; m represents a number of 1 or 2; n represents a number of 0 to 10; B represents an anion selected from -SO3-, -COO-, -OP=O(H)O-, -OP=O(CH3)O-, -OP=O(OR4)O-, and -OP=O(OH)O-; and R4 represents an alkyl group having 1 to 8 carbon atoms which may have a heteroatom.

[0021] In the present invention, a zwitterion is a molecule having both a positive charge and a negative charge within a single molecule. The zwitterion of the present invention has a cationic moiety represented by A and an anionic moiety represented by B. The zwitterion of the present invention may have one or more oxyalkylene groups between the cationic moiety represented by A and the anionic moiety represented by B. By having one or more rotatable oxyalkylene structures between the cationic moiety and the anionic moiety, the cation and the anion can independently assume positions freely, which is thought to lower the melting point and viscosity. Regarding the number of rotatable oxyalkylene structures between the cationic moiety and the anionic moiety, those in which the repeating number n of the oxyethylene structure is 1 to 10 are preferably used, more preferably 1 to 6, and even more preferably 1 to 3.

[0022] In the present invention, the alkyl group refers to a saturated chain hydrocarbon group, and includes a linear alkyl group and a branched alkyl group. Preferably, it is an alkyl group having 1 to 8 carbon atoms, and includes a linear or branched alkyl group having 1 to 8 carbon atoms. More preferably, it is a linear or branched alkyl group having 1 to 6 carbon atoms, and even more preferably, it is a linear or branched alkyl group having 1 to 4 carbon atoms. Specific examples of these alkyl groups include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, and an n-octyl group. In the present invention, the alkenyl group refers to an unsaturated chain hydrocarbon group having one double bond, and includes a linear alkenyl group and a branched alkenyl group. Preferably, it includes a linear or branched alkenyl group having 2 to 8 carbon atoms. More preferred are straight-chain or branched-chain alkenyl groups having 2 to 6 carbon atoms, and more preferred are straight-chain or branched-chain alkenyl groups having 2 to 4 carbon atoms. Specific examples of these alkenyl groups include vinyl, 1-propenyl, 2-propenyl (allyl), butenyl, pentenyl, and hexenyl. In the present invention, the alkylene group is a divalent saturated chain hydrocarbon group, and includes straight-chain and branched-chain alkylene groups. Preferably, it is a straight-chain or branched-chain alkylene group having 1 to 4 carbon atoms, and more preferably, it is a straight-chain or branched-chain alkylene group having 2 to 4 carbon atoms. Specific examples include a methylene group, an ethylene group, a trimethylene group, a propylene group, and a tetramethylene group.

[0023] R 1represents a linear alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, or a C1-8 linear alkyl-(OCH2CH2)m-. The linear alkyl group having 1 to 8 carbon atoms is preferably a linear alkyl group having 1 to 6 carbon atoms, and more preferably a linear alkyl group having 1 to 4 carbon atoms. Specific examples of these alkyl groups include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an isobutyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, and an n-octyl group. Of these, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, and an n-hexyl group are preferred, and a methyl group, an ethyl group, an n-propyl group, and an n-butyl group are more preferred. An alkenyl group having 2 to 8 carbon atoms includes a linear or branched alkenyl group having 2 to 8 carbon atoms. Preferably, it is a linear or branched alkenyl group having 2 to 6 carbon atoms, more preferably a linear or branched alkenyl group having 2 to 4 carbon atoms. Of these, vinyl, propenyl, butenyl, pentenyl, and hexenyl groups are preferred, with vinyl, 1-propenyl, 2-propenyl (allyl), and butenyl being more preferred. The C1-8 linear alkyl group in the group represented by C1-8 linear alkyl-(OCH2CH2)m- is preferably a linear alkyl group having 1 to 6 carbon atoms, more preferably a linear alkyl group having 1 to 4 carbon atoms. Specific examples of these alkyl groups include methyl, ethyl, n-propyl, n-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Of these, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups are preferred, and methyl, ethyl, n-propyl, and n-butyl groups are more preferred.

[0024] R 2 represents an alkylene group having 1 to 4 carbon atoms, including a linear or branched alkylene group having 1 to 4 carbon atoms. Specific examples include a methylene group, an ethylene group, a trimethylene group, a propylene group, and a tetramethylene group.

[0025] R 3Examples of the alkylene group include linear or branched alkylene groups having 2 to 4 carbon atoms, and specific examples thereof include an ethylene group, a trimethylene group, a propylene group, and a tetramethylene group. 3 is preferably an oxyethylene group, an oxytrimethylene group, an oxypropylene group, or an oxytetramethylene group. n may be a number from 0 to 10, preferably a number from 1 to 10, more preferably a number from 1 to 8, even more preferably a number from 1 to 6, and even more preferably a number from 1 to 4. B is the anion part of the zwitterion, and is -SO3 - , -COO - , -OP=O(H)O - , -OP=O(CH3)O - , -OP=O(OR 4 ) O - , and -OP=O(OH)O - represents an anion selected from 4 is an alkyl group having 1 to 8 carbon atoms which may have a heteroatom. 4 As the —COO group, a methyl group, an ethyl group, etc. are preferred. - , -SO3 - , and -OP=O(OH)O - Preferably, the anion is selected from the group consisting of -COO - is preferred.

[0026] A is a cation portion of the zwitterion and represents a cation selected from imidazolium cation, phosphonium cation, ammonium cation, sulfonium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation, morpholinium cation, cyclopropenium cation and piperidinium cation. Among these cations, imidazolium cation, ammonium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation, morpholinium cation or piperidinium cation is preferred, imidazolium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation or piperidinium cation is more preferred, and imidazolium cation is even more preferred. Furthermore, the imidazolium cation, pyrazolium cation, pyridinium cation, pyrrolidinium cation or piperidinium cation can be selected from R 1 or R 2 At least one of the functional groups is substituted on the nitrogen to be cationized, and the cation generated at this time is R 2 It forms a betaine structure with the acidic group of the substituent. Specific examples of cations with such a structure include:

[0027]

[0028] Examples include:

[0029] (Additive (X)) The additive (X) used in the present invention may be two or more types compatible with component (A). Specifically, two or more types may be selected from the following (i) to (Vi). Among (i) to (Vi), two or more types may be selected from the same category. Here, compatibility does not necessarily mean compatibility with component (A), but rather compatibility after addition of a solvent such as water and multiple additives (X). (i) A small molecular compound having no hydroxyl group, (ii) A compound having one or more hydroxyl groups, (iii) A peptide, (iv) A polymer compound, (v) A surfactant, (vi) A zwitterionic polymer or zwitterionic oligomer represented by the following general formula (2):

[0030]

[0031] (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, and are —COO—, —SO—, —OP═O(H)O—, —OP═O(CH)O—, and —OP═O(OR 5 ) O- (wherein R 5 represents a hydrogen atom or a methyl group); Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a straight-chain or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m, and n each represent a number indicating the content ratio of each repeating unit and satisfying the conditions 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s, and t each represent an integer of 0 to 6.

[0032] (i) Small molecular compounds without hydroxyl groups or planar compounds. Suitable small molecular compounds include small molecular compounds with polar functional groups other than hydroxyl groups (preferably compounds with a molecular weight of 120 or less) and planar compounds. Compounds that penetrate cell membranes and exhibit cell permeability are preferred. Dimethyl sulfoxide (DMSO) is particularly cell-permeable, and is thought to penetrate into cells and suppress the crystallization of free water. In the present invention, it has been suggested that the toxicity of DMSO may be reduced by combining it with another additive (X) rather than by using DMSO alone. Other examples include amide compounds such as dimethylformamide, dimethylacetamide, methylacetamide, acetamide, acrylamide, propionamide, methacrylamide, isobutylamide, lactamide, nicotinamide, and isophthalamide; organic solvents such as hexane, benzene, toluene, and β-valerolactone; aromatic (fluorescent dye) molecules such as propidium iodide; and steroid compounds such as cholesterol.

[0033] (ii) Compounds Having One or More Hydroxyl Groups Examples of compounds having one or more hydroxyl groups include monohydric alcohols, polyhydric alcohols, monosaccharides, disaccharides, and oligosaccharides. Examples of monohydric alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, octanol, and phenol. Examples of polyhydric alcohols include dihydric and trihydric polyhydric alcohols, specifically ethylene glycol, propylene glycol, glycerin, diethylene glycol, dipropylene glycol, and diglycerin, with ethylene glycol, propylene glycol, and glycerin being more preferred. Examples of monosaccharides include aldoses and ketoses, which are classified based on the carbonyl group, and trioses, tetroses, pentoses, hexoses, and heptoses, which are classified based on the number of carbon atoms, as well as sugar alcohols. Specific examples include glyceraldehyde, dihydroxyacetone, erythrose, erythrulose, ribose, ribulose, glucose, fructose, and sedoheptulose. Monosaccharides also include sugar alcohols, such as erythritol, sorbitol, and mannitol, which are formed by reducing aldehydes or ketones to alcohols. Specifically, monosaccharides such as glucose and fructose, and sugar alcohols such as erythritol, sorbitol, and mannitol are more preferred. Examples of disaccharides include maltose, cellobiose, lactose, sucrose, isomaltose, and trehalose. Examples of oligosaccharides include those with three or more linked units, such as raffinose, panose, maltotriose, cellotriose, melezitose, and gentianose, and those with four or more linked units, such as stachyose and cellotetraose. Of these polyhydric alcohols and sugars, polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerin, monosaccharides such as glucose and fructose, sugar alcohols such as erythritol, sorbitol, and mannitol, and disaccharides such as maltose, cellobiose, lactose, sucrose, isomaltose, and trehalose are more preferred.It is believed that these polyhydric alcohols and sugars adhere to the outermost cell surface of spheroids and the like, thereby enhancing their protective effect during storage.

[0034] (iii) Peptides or Proteins Peptides are composed of approximately 2 to 50 amino acids linked together, and examples include whey peptides, fish peptides, soybean peptides, sardine peptides, and collagen peptides. It is believed that peptides enhance their protective effect during storage by electrostatically interacting with cells on the surface of spheroids, etc. Antifreeze proteins, polypeptides that have the ability to inhibit ice crystal formation, are also examples.

[0035] (iv) Polymer Compounds Polymer compounds may be natural or non-natural. Natural polymers include starch, glycogen, cellulose, alginic acid, pectin, agarose, carrageenan, and the like. Non-natural polymers include carboxymethyl cellulose, polyvinyl alcohol, and vinyl alcohol oligomers. Cellulose nanofibers are also sometimes used for culture and can be used continuously as a storage medium. They are useful for regulating osmotic pressure and have the function of protecting cells on the surface of spheroids, etc.

[0036] (v) Surfactants Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Nonionic surfactants are preferred. Examples of nonionic surfactants include ester, ether, ester-ether, and fatty acid alkanolamide types. Ester types are characterized by an ester bond between a polyhydric alcohol and a fatty acid, and examples include glycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters. Examples of ether types include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, and polyoxyethylene polyoxypropylene glycols. Ester-ether types are esters of polyhydric alcohols and fatty acids, with ethylene oxide added to introduce polyethylene ether side chains. All of these types regulate osmotic pressure and protect the cells in the outermost layer of the spheroids.

[0037] (vi) Zwitterionic polymer or zwitterionic oligomer represented by the general formula (2) The zwitterionic polymer (2) has an aprotic zwitterion, i.e., a cationic moiety and an anionic moiety, in the side chain of at least 1 repeating unit. The zwitterionic structure is represented by X in the general formula (2). 1 and a heterocyclic cation containing a nitrogen atom (N) and Y 1 In addition, the side chain of m repeating units is formed with an anion of X 2 and a heterocyclic cation containing a nitrogen atom (N) and Y 2 and an anion of the formula (I). In the present invention, the term "zwitterionic structure" refers to a compound (internal salt) that has a positive charge and a negative charge at non-adjacent positions within the same molecule, no dissociable hydrogen atoms are bonded to the positively charged atom, and the molecule as a whole has no charge. Thus, by having zwitterions in the side chains of the repeating units of zwitterionic polymer (2), it is believed that the polymer exhibits a protective effect on cell membranes and functions as a non-cryopreservative. Furthermore, by using zwitterionic polymer (2) in combination with one or more additives selected from (i) to (v), the polymer changes from an associated state due to the zwitterions in the side chains to a dispersed state, which is believed to affect cell membranes and exhibit non-cryopreservative properties.

[0038] In general formula (2), l, m, and n are numbers that respectively indicate the content ratio of each repeating unit, and are numbers that satisfy the following conditions: 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1. When m=n=0, general formula (2) represents a homopolymer having a zwitterionic functional group in the side chain. When m is a number greater than 0 and n=0, general formula (2) represents a random or block copolymer having two types of zwitterionic structures. When m=0 and n is a number greater than 0, general formula (2) represents a random or block copolymer having a repeating unit having a zwitterionic functional group in the side chain and a repeating unit having Z in the side chain. When m and n are numbers greater than 0, general formula (2) represents a ternary random or block copolymer. p, q, and t each independently represent an integer from 0 to 6. Specifically, this moiety is a single bond or a linear alkylene group having 1 to 6 carbon atoms, and a single bond, a methylene group, an ethylene group, a trimethylene group, or a tetramethylene group is preferred. Furthermore, from the viewpoint of cryoprotection of biological samples, this moiety is more preferably a single bond, a methylene group, or an ethylene group, and even more preferably a single bond or a methylene group. p, q, and t may be the same or different. r and s each independently represent an integer of 0 to 6, and an integer of 1 to 6 is preferred. This moiety is preferably a linear alkylene group having 1 to 6 carbon atoms, and specifically, a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, or a hexamethylene group is preferred. r and s may be the same or different. In general formula (2), X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom. 1 and heterocyclic cations containing a nitrogen atom (N), such as imidazolium cations (X 1 = N, e = 0), pyridinium cation (X 1 = C, e = 1), pyrrolinium cation (X 1 = C, e = 0), pyrazinium cation (X 1 Among these, imidazolium cations (X = N, e = 1) are mentioned. 1 = N, e = 0), pyridinium cation (X 1In the general formula (1), X is preferably C, and e is preferably 1. 2 and heterocyclic cations containing a nitrogen atom (N), such as imidazolium cations (X 2 = N, f = 0), pyridinium cation (X 2 = C, f = 1), pyrrolinium cation (X 2 = C, f = 0), pyrazinium cation (X 2 Among these, imidazolium cations (X = N, f = 1) are mentioned. 2 = N, f = 0), pyridinium cation (X 2 = C, f = 1) is preferred. 1 and Y 2 may be the same or different, -COO - , -SO3 - , -OP=O(H)O - , -OP=O(CH3)O - and -OP=O(OR 1 ) O - represents an anion selected from the group consisting of: 1 and Y 2 As for -COO - , -SO3 - and -OP=O(OH)O - Preferably, the anion is selected from the group consisting of -COO - and -SO3 - More preferred are anions selected from -COO -Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a linear or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain. Specifically, examples thereof include a hydrogen atom, a phenyl group, a naphthyl group, a C1 to C22 alkyl-substituted phenyl group, a C1 to C22 alkyl-substituted naphthyl group, an imidazolyl group, a triazolyl group, a pyridyl group, a pyrrolyl group, a pyrazinyl group, a furyl group, a thienyl group, an oxazolyl group, a thiazolyl group, pyridinium chloride, a C1 to C22 alkylpyridinium chloride, imidazolinium chloride, a C1 to C22 alkylimidazolinium chloride, pyridinium bromide, a C1 to C22 alkylpyridinium bromide, imidazolinium bromide, a C1 to C22 alkylimidazolinium bromide, pyrrolidinium chloride, C1 to C Examples of the alkyl group include C1 to C22 alkylpyrrolidinium chloride, pyrrolidinium bromide, C1 to C22 alkylpyrrolidinium bromide, tetramethylammonium chloride, C2 to C22 alkyltrimethylammonium chloride, tetramethylammonium bromide, C2 to C22 alkyltrimethylammonium bromide, tetraphenylphosphonium chloride, tetraphenylphosphonium bromide, trimethylsulfonium chloride, trimethylsulfonium bromide, C1 to C22 straight-chain or branched-chain alkyl groups, ethoxyethyl groups, ethoxyethoxyethyl groups, and ethoxyethoxyethoxyethyl groups.Preferred examples of Z include a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group or a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, and more preferred examples include an imidazolyl group, a triazolyl group, a pyridyl group, a pyrrolyl group, a pyrazinyl group, a furyl group, a thienyl group, an oxazolyl group, a thiazolyl group, pyridinium chloride, C1 to C22 alkylpyridinium chloride, imidazolinium chloride, C1 to C22 alkylimidazolinium chloride, pyridinium bromide, C1 to C22 alkylpyridinium bromide, imidazolinium bromide, C1 to C22 alkylimidazolinium bromide, pyrrolidinium chloride, C1 to C22 alkylpyrrolidinium chloride, pyrrolidinium bromide, and C1 to C22 alkylpyrrolidinium bromide. More preferred examples include an imidazolyl group, a pyridyl group, a pyridinium chloride, a C1-C22 alkylpyridinium chloride, an imidazolinium chloride, a C1-C22 alkylimidazolinium chloride, a pyridinium bromide, a C1-C22 alkylpyridinium bromide, an imidazolinium bromide, and a C1-C22 alkylimidazolinium bromide. The molecular weight of the zwitterionic polymer (2) must be determined based on the main chain structure, the side chain structure, and the proportion of each to the total, and known measurement methods that reflect the respective characteristics can be used. Furthermore, in the case of zwitterionic polymers, it is known that the main chain conformation changes depending on the type of zwitterion, pH, and the concentration of electrolytes such as NaCl.

[0039] The main chain structure of the zwitterionic polymer (2) may be star-shaped, comb-shaped, crosslinked, or the like. Any of these structures can be used for the purposes of the present invention, but the main chain conformation varies depending on the type of side chain. In the case of a polymer with a linear main chain, dispersibility in water can be improved by expanding the polymer through the addition of an electrolyte such as NaCl. Furthermore, the molecular weight of the zwitterionic polymer (2) is preferably a certain size, since the zwitterionic structure present in the side chain affects cell membrane structure and cell permeability, the formation of extracellular ice crystals, and the solubility of poorly soluble substances. When the molecular chain is expanded by an electrolyte, the molecular weight, in terms of polyethylene oxide, is preferably 10,000 or more and the Mn is preferably 5,000 or more, and more preferably 10,000 to 2,000,000 and 5,000 to 1,500,000. Furthermore, the molecular weight distribution of the polymer is also taken into consideration.

[0040] Zwitterionic polymer (2) does not penetrate cells, but instead forms a matrix outside the cell and accumulates around the cell membrane outside the cell. One possible reason for this is that the zwitterionic molecule has a betaine structure, which means it is electrically charged and therefore unable to penetrate the cell membrane as a zwitterionic polymer. Furthermore, if the polymer side chain contains a hydrophobic functional group with high affinity for the cell membrane, and this functional group is inserted into the cell membrane from the outside of the cell, the hydrophilic polymer is tethered to the cell membrane outside the cell, and the polymer accumulates around the cell membrane outside the cell. Considering these properties in the context of multicellular aggregates such as spheroids, it is expected that the polymer will protect the cell aggregate by protecting the outermost layer of cells. Examples of functional groups that contribute to such accumulation include those containing an alkylene group between the cation and anion in general formula (2). It is also preferable that Z contains a C1-C22 alkyl group, or preferably a C3-C18 alkyl group. The ratio of such substituents to the entire polymer is preferably 0.001 to 10 mol %, more preferably 0.01 to 1 mol %, and even more preferably 0.1 to 0.5 mol %.

[0041] The additive (X) is used by selecting two or more kinds from the additives (i) to (vi) described above. Preferred combinations include combinations of two or more selected from (i) (e.g., a combination of DMSO and dimethylacetamide, a combination of DMSO and acetamide, a combination of DMSO and dimethylformamide, etc.); combinations of two or more selected from (ii) (e.g., a combination of methanol and propylene glycol, a combination of glycerol and propylene glycol, a combination of ethanol, ethylene glycol and sucrose, etc.); combinations of one or more selected from (i) and one or more selected from (ii) to (vi); combinations of one or more selected from (ii) and one or more selected from (i), (iii) to (vi); combinations of one or more selected from (i), one or more selected from (ii), and one or more selected from (iii) to (vi) (e.g., a combination of DMSO, acetamide, propylene glycol, polyvinyl alcohol, antifreeze protein and polyZI, etc.).More specifically, (1) a combination of DMSO and one or more hydroxyl group-containing compounds selected from polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerin, monosaccharides such as glucose and fructose, sugar alcohols such as erythritol, sorbitol, and mannitol, and disaccharides such as maltose, cellobiose, lactose, sucrose, isomaltose, and trehalose; (2) a combination of DMSO and one or more hydroxyl group-containing compounds selected from polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerin, monosaccharides such as glucose and fructose, sugar alcohols such as erythritol, sorbitol, and mannitol, and disaccharides such as maltose, cellobiose, lactose, sucrose, isomaltose, and trehalose. (3) a combination of one or more hydroxyl group-containing compounds selected from polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerin, monosaccharides such as glucose and fructose, sugar alcohols such as erythritol, sorbitol, and mannitol, and disaccharides such as maltose, cellobiose, lactose, sucrose, isomaltose, and trehalose, and one or more hydroxyl group-containing compounds selected from peptides, polymeric compounds, surfactants, and zwitterionic polymers (2); and (4) a combination of DMSO, dimethylacetamide, and ethylene glycol or propylene glycol.

[0042] (Composition) The composition for non-cryopreservation of multicellular populations of the present invention contains one or more aprotic zwitterionic compounds (A) and two or more additives (X) that are compatible with a solution containing component (A). Here, "compatible" does not necessarily mean that the compound is compatible with component (A); it may be compatible after the addition of a solvent such as water and multiple additives (X). When used for non-cryopreservation, the aprotic zwitterionic compound (A) and the additives (X) are preferably used in a mass ratio of 1:0.1 to 1:50, more preferably 1:0.5 to 1:5, and even more preferably 1:1 to 1:4. Furthermore, the composition of the present invention may contain water, a cell culture medium, and physiological saline components in addition to the aprotic zwitterionic compound (A) and two or more additives (X). In this case, the composition of the present invention is preferably used in a ratio of aprotic zwitterionic compound (A):water of 0.1:10 to 4:10, more preferably 0.5:10 to 3:10, and even more preferably 0.5:10 to 2:10, by mass. The aprotic zwitterionic compound (A) increases the antifreeze properties at low temperatures by mixing with water throughout the medium, while the additive (X) is thought to regulate osmotic pressure, protect the entire spheroid, and, if it penetrates into the cells, increase the antifreeze properties of the free water within the cells. However, in the case of spheroids and the like, although the cells on the surface of the aggregates are in direct contact with the medium, the extracellular matrix generated by the formation of spheroids acts as a barrier, so it is unclear whether the additives necessarily penetrate to the internal cells. However, some aprotic zwitterionic compounds (A) and additives (X) are thought to be useful in preventing the freezing of the free water within the intercellular matrix. However, since high molecular weight additives (X) cannot penetrate between cells as desired, they are thought to play a role in protecting the spheroid as a whole and regulating osmotic pressure. From this perspective, when selecting two or more additives (X), it is preferable to select additives that are thought to have different functions. Therefore, the combination of two or more additives (X) is preferably the above-mentioned combination.

[0043] When applying iPS cell spheroids differentiated into spheroids or differentiated iPS cell spheroids to regenerative medicine, since the spheroids are produced in different locations and transported to different sites for regenerative medicine, it is important that the water in the medium does not crystallize when stimulated, even during cryogenic storage. Possible conditions for this include having no melting point or glass transition point above the storage temperature, and ensuring that the medium is not supercooled. The melting point and glass transition point can be determined by differential scanning calorimetry (DSC). Furthermore, supercooling can be determined by observing the medium at the storage temperature while stimulating it to determine whether crystallization occurs.

[0044] Another aspect of the present invention is a method for preserving a multicellular population under non-freezing conditions using the non-cryopreservation composition of the present invention. The subject of non-cryopreservation in the method of the present invention is a multicellular population. As described above, multicellular populations include spheroids, organoids, cell sheets, tissue structures, organ slices, organs, and living organisms / individuals. However, spheroids, organoids, cell sheets, and cell structures are preferred, with spheroids being more preferred. Specifically, a multicellular population is added to the composition of the present invention and then preserved under non-freezing conditions. Non-cryopreservation is performed in a storage space between liquid nitrogen temperature and 37°C, preferably between -85°C and room temperature, and more preferably between -50°C and 0°C. Considering the utilization of the food cold chain, it is preferable to maintain the multicellular population under non-freezing conditions between -85°C and 4°C, preferably between -30°C and -4°C, and more preferably between -30°C and -15°C.

[0045] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0046] (Substance to be Evaluated) The aprotic zwitterion used in the present invention can be produced with reference to J. Am. Chem. Soc. 139, 16052-16055 (2017).

[0047] (Synthesis Example 1) OE2imC3C

[0048]

[0049] 15.7 g (656 mmol) of NaH (Kanto Chemical Co., Inc.) was suspended in 50 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries, Ltd.), and 13.8 g (202 mmol) of imidazole (Tokyo Chemical Industry Co., Ltd.) was added, followed by stirring at room temperature for 24 hours. 37.0 g (202 mmol) of 1-bromo-2-(2-methoxyethoxy)ethane (Tokyo Chemical Industry Co., Ltd.) was added, followed by stirring at 70°C for 6 hours. The white precipitate was filtered off, and the filtrate was concentrated and then distilled (125°C, 1 mmHg) to obtain 1-(2-(2-methoxyethoxy)ethyl)imidazole. This was dissolved in 250 mL of acetonitrile, and 29 g (150 mmol) of ethyl 4-bromobutyrate was added. The mixture was refluxed at 80°C for 16 hours. After washing with diethyl ether, the mixture was applied to a column of anion exchange resin (Amberlite IRN 78A), and the eluate was evaporated under reduced pressure to obtain OE2imC3C. NMR data: δ=2.13-2.27 (4H, m, CH2CO and CH2CH2CO), 3.37 (3H, s, CH3O), 3.51-3.65 (4H, m, CH3OCH2CH2), 3.86 (2H, t, J = 3.6Hz, OC H2CH2N), 4.40 (2H, t, J = 6.7Hz, NCH2CH2CH2COO), 4.66 (2H, t, J = 3.7Hz, OCH2CH2N), 7.29 and 7.49 (2H, t, J = both 1.6Hz, NCHCHN), 11.00 (1H, s, NCHN). 13C NMR (100MHz; CDCl3; Me4Si) δ=27.20 and 34.30 (NCH2CH2CH2COO), 48.94 (OCH2CH2N), 49.47 (NCH2CH2CH2COO), 58.65 (CH3O), 69.19 (OCH2CH2N), 69.93 and 71.29 (OCH2CH2O), 121.22 and 122.58 (NCHCHN), 138.73 (NCHN), 176.63 (CH2COO). Elemental analysis: OE2imC3C・2.5H2O (Found: C, 48.0; H, 8.4; N, 9.3. Calc. for C 12 H 25 N2O 6.5 :C, 47.8; H, 8.4; N, 9.3%).

[0050] (Synthesis example 2) C1imC3C

[0051]

[0052] 22.2 g (270 mmol) of 1-methylimidazole and (53.7 g: 270 mmol) of ethyl 4-bromobutyrate were dissolved in 20 mL of acetonitrile and stirred at 50°C for 5 hours. After removing the precipitate, the mixture was dried under reduced pressure, washed three times with diethyl ether, and dried under reduced pressure. The residue was subjected to anion exchange resin to obtain C1imC3C. １ H NMR (400MHz; DMSO-d6; Me4Si) δ = 1.77 (2H, t, J = 6.4Hz, CH2CO), 1.86 (2H, J = 7.3 Hz, quin, CH2CH2CO), 3.82 (3H, s, CH3N), 4.13 (2H, t, J=6.8Hz, NCH2CH2), 7.66 and 7.76 (2H, t, J=both 1.6Hz, NCHCHN), 9.50 (1H, s, NCHN). 13C NMR (100MHz; DMSO-d6; Me4Si) δ = 27.80 (CH2CH2CO), 35.36 (CH2CO), 36.10 (CH3N), 49.61 (NCH2CH2), 122.91 and 123.89 (NCHCHN), 137.700 (NCHN), 174.02 (CH2COO).

[0053] (Synthesis example 3) C1imC3S

[0054]

[0055] C1imC3S was obtained in the same manner as in Synthesis Example 2, except that 1,3-propane sultone was used instead of ethyl 4-bromobutyrate.

[0056] (Synthesis example 4) C4imC3S

[0057]

[0058] C4imC3S was obtained by the same procedure as in Synthesis Example 2, except that 4-butylimidazole was used instead of 1-methylimidazole and 1,3-propanesultone was used instead of ethyl 4-bromobutyrate.

[0059] (Synthesis example 5) VimC3C

[0060]

[0061] VimC3C was obtained in the same manner as in Synthesis Example 2, except that vinylimidazole (Tokyo Chemical Industry Co., Ltd.) was used instead of 1-methylimidazole.

[0062] (Synthesis Example 6)

[0063]

[0064] To 10 mL of pure water, 3 g (0.014 mmol) of VimC3C was added, and 22.83 g (0.0014 mmol) of 2,2'-azobis(isobutyronitrile) (Tokyo Chemical Industry Co., Ltd.) was added as a polymerization initiator, followed by stirring at 80°C for 16 hours. The mixture was dialyzed against pure water and dried under reduced pressure to obtain Poly(VimC3C).

[0065] Synthesis Example 7 (C1imC2OE3C(R 1 = CH3, A = imidazolium cation, R 2 = CH2CH2, R 3 =CH2CH2, n=3, B=-COO -) [Raw Materials] Benzenesulfonyl chloride, p-toluenesulfonyl chloride, 1-methylimidazole, sodium hydroxide, and diethyl ether were purchased from Tokyo Chemical Industry Co., Ltd. Toluene, dichloromethane, MeOH, chloroform-d1, 99.8 atom% D with 0.03 vol% TMS were purchased from Kanto Chemical Co., Inc. Tetrahydrofuran with stabilizer and hydrochloric acid were purchased from FUJIFILM Wako Pure Chemical Corporation. Aluminum oxide (active, basic, Brockmann 1) was purchased from Sigma-Aldrich. Amberlite IRN-78, ion exchange resin, nuclear grade, was purchased from Alfa Aesar. tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was purchased from Accela ChemBio.

[0066] (1) Synthesis of p-toluenesulfonyl PEG4 tert-butyl ester (1a) 4.9 g of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was placed in a 50 mL recovery flask and dissolved in 30 mL of dichloromethane (DCM). The recovery flask was then cooled on ice, and 2.45 g of NaOH was added to the recovery flask to make a 3.5 molar equivalent of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate solution, followed by stirring under ice cooling. Furthermore, 4.4 g of p-toluenesulfonyl chloride was added dropwise to the stirred eggplant flask while cooling with ice so that the ratio was 1.3 equiv. relative to tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate. After the entire amount was added dropwise, the mixture was stirred at room temperature for 28 hours to allow the reaction to proceed. After stirring for 28 hours, solid impurities were removed using filter paper (5C, ADVANTEC). The reaction solution containing the filtered product was placed in a separatory funnel and washed with 60 mL of DCM. Water was then added, and the pH and sodium concentration of the aqueous layer after separation were measured. Separation was continued until the pH reached 7 and the measured sodium concentration reached 0 ppm. After separation, the DCM layer was dried under reduced pressure to obtain the product. The structure of the product was confirmed by 1H NMR, and the product was used as the next raw material without purification.

[0067] (2) Synthesis of p-toluenesulfonyl PEG4 acid (2a) The synthesized p-toluenesulfonyl PEG4 tert-butyl ester was placed in a 200 mL recovery flask, and 45 g of 36% HCl aqueous solution was added to the p-toluenesulfonyl PEG4 tert-butyl ester to give a 25% equiv. solution. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. After stirring for 3 hours, the reaction solution containing the product was placed in a separatory funnel and washed with 120 mL of DCM. Water was then added, and the mixture was separated until the pH of the aqueous layer reached 7. After separation, the DCM layer was dried under reduced pressure to obtain the product. The structure of the product was confirmed by 1H NMR, and the product was used as the next raw material without purification.

[0068] (3) Synthesis of imidazolium p-toluenesulfonyl PEG4 acid (3a) The synthesized p-toluenesulfonyl PEG4 acid was placed in a 200 mL recovery flask, and 20 mL of tetrahydrofuran purified with aluminum oxide (active, basic, Brockmann 1) was added and dissolved. The recovery flask was then ice-cooled, and 1.77 g of 1-methylimidazole was added. After ice-cooling and stirring, the mixture was refluxed at 70 °C for 45 hours using a reflux condenser to allow the reaction to proceed. After refluxing for 45 hours, the reaction solution was dried under reduced pressure, and the resulting product was washed with diethyl ether (40 mL, 4 h, 3 times) in a 200 mL recovery flask. After washing, the product was obtained by drying under reduced pressure. The structure of the product was confirmed by 1H NMR, and the product was used as the raw material for the next step without purification.

[0069] (4) Synthesis of imidazolium hydroxide (4a) and C1imCOE3C. The synthesized imidazolium p-toluenesulfonate was dissolved in 600 mL of HO / MeOH (1 / 2: w / w) solvent and transferred to a 1 L bottle. Then, 35 mL of anion exchange resin (Amberlite IRN-78, exchange capacity 1.1 eq / L) was added, and the mixture was stirred at room temperature for 10 days to produce imidazolium hydroxide. After anion exchange, the anion exchange resin was removed using filter paper (5C, ADVANTEC), and the reaction solution was transferred to a 20 mL recovery flask. The resulting reaction solution was concentrated, and the neutralization reaction and the C1imCOE3C production reaction were allowed to proceed. Then, diethyl ether was added to the 20 mL recovery flask containing the product and washed (20 mL, 1 day, 3 times). After washing, the product was dried under reduced pressure to obtain an oily product (purity 98%). The yield based on tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was 56%.

[0070] 1H NMR (400 MHz; CDCl3; Me4Si) δ = 2.424 (2H, t, J = 4.0 Hz, CH2COO), 3.538 - 3.644 (8H, m, OCH2CH2OCH2CH2OCH2CH2COO), 3.790 (2H, t, J = 11.6 Hz, NCH2CH2OCH2), 3.919 (2H, t, J = 9.6 Hz, NCH2CH2OCH2), 3.980 (3H, s, CH3N), 4.499 (2H, t, J = 9.6 Hz, NCH2CH2O), 7.238 and 7.243 (1H, m, NCHCHN), 10.465 (1H, s, NCHN). Mass spectrometry with fast atom bombardment ionization: in positive mode, m / z = 287.1610 (M + H + , found) and 287.1607 (M + H + , calculated); and in negative mode, m / z = 285.1452 (M - H + , found) and 285.1450 (M - H + )(calculated).

[0071] Synthesis Example 8 (OE2imC2OE3C(R 1 = H3C(OCH2CH2)2, A = imidazolium cation, R 2 = CH2CH2, R 3 = CH2CH2, n = 3, B = -COO -) [Raw Materials] Benzenesulfonyl chloride, p-toluenesulfonyl chloride, imidazole, diethylene glycol monomethyl ether, sodium hydroxide, and diethyl ether were purchased from Tokyo Chemical Industry Co., Ltd. Toluene, dichloromethane, MeOH, chloroform-d1, 99.8 atom% D with 0.03 vol% TMS were purchased from Kanto Chemical Co., Inc. Tetrahydrofuran with stabilizer and hydrochloric acid were purchased from FUJIFILM Wako Pure Chemical Corporation. Aluminum oxide (active, basic, Brockmann 1) was purchased from Sigma-Aldrich. Amberlite IRN-78, ion exchange resin, nuclear grade was purchased from Alfa Aesar. tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was purchased from Angene International Limited.

[0072] (1) Synthesis of p-toluenesulfonyl PEG4 tert-butyl ester (1a) 3.72 g of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was placed in a 50 mL recovery flask and dissolved in 10 mL of DCM. The recovery flask was then cooled on ice, and 3.38 g of NaOH was added to the recovery flask to a molar equivalent of 6.2 equivalents relative to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate, followed by stirring under ice cooling. Furthermore, 1.3 equivalents relative to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was added. 3.3 g of p-toluenesulfonyl chloride was added dropwise to a stirring recovery flask under ice cooling so that the temperature reached 100°C. After the entire amount was added dropwise, the mixture was stirred at room temperature for 17 hours to allow the reaction to proceed. After stirring for 17 hours, solid impurities were removed using filter paper (5C, ADVANTEC). The reaction solution containing the filtered product was placed in a separatory funnel and washed with 60 mL of DCM. Water was then added, and the pH and sodium concentration of the aqueous layer after separation were measured. Separation was continued until the pH reached 6 and the measured sodium concentration reached 0 ppm. After separation, the DCM layer was dried under reduced pressure to obtain the product. The structure of the product was confirmed by 1H NMR and used in the next reaction.

[0073] (2) Synthesis of p-toluenesulfonyl PEG4 acid (2a) The synthesized p-toluenesulfonyl PEG4 tert-butyl ester was placed in a 200 mL recovery flask, and 34.4 g of 36% HCl aqueous solution was added so that the concentration was 25 equiv. relative to the p-toluenesulfonyl PEG4 tert-butyl ester. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. After stirring for 3 hours, the reaction solution containing the product was placed in a separatory funnel and washed with 50 mL of DCM. Water was then added, and the mixture was separated until the pH of the aqueous layer after separation reached 7. After separation, the DCM layer was dried under reduced pressure to obtain the product. The structure of the product was confirmed by 1H NMR and used in the next reaction.

[0074] Synthesis of OE2im: This was prepared with reference to Cellulose, 29, 3017-3024. To synthesize diethylene glycol monomethyl benzenesulfonate, sodium hydroxide (2.5 equivalents, 55 g, 1.38 mol) was mixed with an equal amount of water (55 g). The resulting solution was added to a solution of diethylene glycol monomethyl ether (1 equivalent, 60 g, 0.5 mol) in toluene (1000 mL), followed by the addition of diethylene glycol monomethyl ether. A catalytic amount of benzyltrimethylammonium hydroxide (10 mL) was added, and the mixture was stirred in an ice bath. Then, benzenesulfonyl chloride (97 g, 0.6 mol) was added dropwise with stirring, and the mixture was refluxed at 70°C for 6 hours. After evaporating the toluene, the resulting solution was dissolved in dichloromethane, washed once with water (3000 mL), and dried over sodium sulfate to obtain the product. To synthesize 1-(2-(2-methoxyethoxy)ethyl)imidazole (OE2im), an aqueous solution of sodium hydroxide was prepared using sodium hydroxide (2.5 equivalents, 56.4 g, 1.4 mol) and an equal volume of water (57 g). This base was added to a solution of imidazole (1 equivalent, 32 g, 0.5 mol) in toluene (1,000 mL), followed by the addition of a catalytic amount of benzyltrimethylammonium hydroxide (10 mL). Diethylene glycol monomethylbenzenesulfonate (123 g, 0.5 mol) was then slowly added while cooling in an ice bath, and the mixture was heated at 70°C for 6 hours to obtain a colorless viscous liquid. After evaporating the toluene, the mixture was distilled under reduced pressure (1 Pa) at 160°C to obtain OE2im (yield: 92%). The structure of the product was confirmed by 1H NMR and used in the subsequent reaction.

[0075] (3) Synthesis of oligoether imidazolium p-toluenesulfonyl PEG4 acid (3b) The synthesized p-toluenesulfonyl PEG4 acid was placed in a 200 mL recovery flask, and 40 mL of toluene was added to dissolve the mixture. Then, 2.3 g of OE2im was added and stirred, followed by refluxing at 80°C for 27 hours using a reflux condenser. After refluxing for 27 hours, the reaction solution was dried under reduced pressure, and the resulting product was washed with diethyl ether (50 mL, 1 day, 3 times) in a 200 mL recovery flask. After washing, the product was dried under reduced pressure to obtain the product. The structure of the product was confirmed by 1H NMR and used in the next reaction.

[0076] (4) Synthesis of oligoether imidazolium hydroxide (4b) and OE2imC2OE3C. The synthesized imidazolium p-toluenesulfonate was dissolved in 400 mL of HO / MeOH (1 / 3: w / w) solvent and transferred to a 1 L bottle. Then, 27 mL of excess anion exchange resin (Amberlite IRN-78, exchange capacity 1.1 eq / L) was added and stirred at room temperature for 10 days to produce oligoether imidazolium hydroxide. Finally, OE2imC2OE3C was synthesized. After anion exchange, the anion exchange resin was removed using filter paper (5C, ADVANTEC), and the reaction solution was transferred to a 50 mL recovery flask. The resulting reaction solution was concentrated, and the neutralization reaction and the OE2imC2OE3C production reaction were allowed to proceed. Then, diethyl ether was added to the 50 mL recovery flask containing the product and washed (30 mL, 1 day, 3 times). After washing, the product was dried under reduced pressure to obtain a liquid product. The purity was 98%, and the yield was approximately 50% based on tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate.

[0077] 1 ​1H NMR (400 MHz; CDCl3; Me4Si) δ = 2.428 (2H, t, J = 10.8 Hz, CH2COO), 3.354 (3H, s, CH3OCH2CH2OCH2CH2N), 3.498 - 3.654 (6H, m, CH3OCH2CH2OCH2CH2N and NCH2CH2OCH2CH2OCH2CH2OCH2CH2COO), 3.782 (2H, t, J = 11.6 Hz, CH3OCH2CH2OCH2CH2N), 3.875 (2H, t, J = 9.6 Hz, NCH2CH2OCH2CH2OCH2CH2OCH2CH2COO), 4.474 (2H, t, J = 9.6 Hz, NCH2CH2OCH2CH2OCH2CH2OCH2CH2COO), 4.563 (2H, t, J = 9.2 Hz, NCH2CH2OCH2CH2OCH2CH2OCH2CH2COO), 7.242 and 7.395 (1H, m, NCHCHN), 10.659 (1H, s, NCHN). Mass spectrometry with fast atom bombardment ionization: in positive mode m / z = 375.2129 (M+H + 、found) and 375.2131 (calculated); and in negative mode, m / z = 373.1977 (M−H + found) and 373.1975 (calculated).

[0078] Synthesis Example 9 (C2imOE3C(R 1 =C2H5, A = imidazolium cation, R 2 =CH2CH2, R 3 =CH2CH2, n = 3, B = -COO -) [Raw Materials] tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was purchased from Angene. p-toluenesulfonyl chloride, sodium hydroxide, and diethyl ether were purchased from Tokyo Chemical Industry Co., Ltd. 1-Ethylimidazole, toluene, dichloromethane, methanol, chloroform-d1, 99.8 atom% D with 0.03 vol% TMS, and dimethyl sulfoxide-d6, 99.9 atom% D with 0.03 vol% TMS were purchased from Kanto Chemical Co., Inc. Hydrochloric acid was purchased from FUJIFILM Wako Pure Chemical Corporation. Amberlite IRN-78, ion exchange resin, nuclear grade, was purchased from Alfa Aesar.

[0079] (1) Synthesis of p-toluenesulfonyl PEG4 tert-butyl ester (1a) 3.7 g of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was placed in a 50 mL recovery flask and dissolved in 40 mL of dichloromethane (DCM). Then, 3.8 g of NaOH (6 equiv. (molar equivalent) relative to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate) was added. The recovery flask was then cooled on ice, and 1.5 equiv. of NaOH relative to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was added. 5.0 g of p-toluenesulfonyl chloride was added dropwise little by little. After the entire amount was added dropwise, the mixture was stirred at room temperature for 34 hours to react, and solid impurities were removed using filter paper (5C, ADVANTEC). The reaction solution containing the filtered product was placed in a separatory funnel. Water was then added, and the sodium concentration of the aqueous layer was measured using a sodium concentration meter. Separation was continued until the measured sodium concentration reached 0 ppm. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 tert-butyl ester (1a) (properties: liquid).

[0080] (2) Synthesis of p-toluenesulfonyl PEG4 acid (2a) p-toluenesulfonyl PEG4 tert-butyl ester was placed in a 200 mL recovery flask, and 9.0 g of 36% aqueous HCl was added in excess of the p-toluenesulfonyl PEG4 tert-butyl ester. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. The reaction solution containing the product was dissolved in DCM and poured into a separatory funnel. Water was then added, the pH was measured using pH test paper, and separation was performed until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure and transferred to a 300 mL recovery flask. 100 mL of excess water was added, and the mixture was stirred at 60°C for 1 hour to decompose unreacted p-toluenesulfonyl chloride. The reaction solution was dissolved in DCM, placed in a separatory funnel, and water was added. The pH was measured using pH test paper, and separation was carried out until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 acid (2a) (state: liquid, yield: 20% based on the initial amount of raw materials charged).

[0081] (3) Synthesis of ethylimidazolium p-toluenesulfonyl PEG4 acid (3a) was placed in a 50 mL recovery flask and dissolved in 10 mL of toluene. The recovery flask was then cooled on ice, and 0.27 g of 1-ethylimidazole (1.05 equiv. relative to the p-toluenesulfonyl PEG4 acid) was added dropwise in small amounts. After the entire amount was added dropwise, the mixture was reacted at 80°C for 20 hours using a reflux condenser, resulting in layer separation into an upper layer containing unreacted material and a lower layer containing the product. The upper layer was discarded by decantation, and the lower layer containing the product was washed with diethyl ether (20 mL, 1 h, 3 times). After washing, the product was dried under reduced pressure to obtain ethylimidazolium p-toluenesulfonate (3a) (properties: liquid).

[0082] (4) Synthesis of ethylimidazolium hydroxide (4a) and C2imOE3C Ethylimidazolium p-toluenesulfonate was placed in a 110 mL vial and dissolved in 70 mL of methanol. Furthermore, 9 mL of strong anion exchange resin Amberlite IRN-78 (exchange capacity 1.1 eq. / L) was added and stirred at room temperature for 5 days to produce ethylimidazolium hydroxide (4a). The reaction solution was concentrated into a 50 mL flask, and the neutralization reaction was allowed to proceed. Then, diethyl ether was added to the recovery flask containing the product and washed (20 mL, 30 min, 3 times). After washing, the product was dried under reduced pressure to obtain C2imOE3C (properties: liquid).

[0083] C2imOE3C: 1 H NMR (400MHz; DMSO; Me4Si) δ = 1.380 (3H, t, J = 7.4Hz, NCH2CH3), 1.970 (2H, t, J = 7. 0Hz, CH2COO), 3.325-3.497 (10H, m, NCH2H2OCH2CH2OCH2CH2OCH2CH2COO), 3.750 and 4.320 (2H, t, J = both 5.0Hz, NCH2CH2OCH2), 4.199 (2H, q, NCH2CH3), 7.747 and 7.826 (1H, m, NCHCHN), 9.613 (1H, s, NCHN).

[0084] Synthesis Example 10 (AimOE3C ((R 1 = -CH2-CH=CH2, A = imidazolium cation, R 2 = CH2CH2, R 3 =CH2CH2, n=3, B=-COO -) ) Synthesis of) [Raw Materials] tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was purchased from Angene. p-toluenesulfonyl chloride, sodium hydroxide, and diethyl ether were purchased from Tokyo Chemical Industry Co., Ltd. 1-Allylimidazole, toluene, dichloromethane, methanol, chloroform-d1, 99.8 atom% D with 0.03 vol% TMS, and dimethyl sulfoxide-d6, 99.9 atom% D with 0.03 vol% TMS were purchased from Kanto Chemical Co., Inc. Hydrochloric acid was purchased from FUJIFILM Wako Pure Chemical Corporation. Amberlite IRN-78, ion exchange resin, nuclear grade, was purchased from Alfa Aesar.

[0085] (1) Synthesis of p-toluenesulfonyl PEG4 tert-butyl ester (1a) 3.0 g of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was placed in a 50 mL recovery flask and dissolved in 30 mL of DCM. Then, 1.6 g of NaOH (3.7 equiv. / molar equivalent) was added to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate. The recovery flask was then cooled on ice, and 1.3 equiv. / mol. of NaOH was added to the recovery flask. 2.6 g of p-toluenesulfonyl chloride was added dropwise little by little. After the entire amount was added dropwise, the mixture was stirred at room temperature for 24 hours to react, and solid impurities were removed using filter paper (5C, ADVANTEC). The reaction solution containing the filtered product was placed in a separatory funnel. Water was then added, and the sodium concentration of the aqueous layer was measured using a sodium concentration meter. Separation was continued until the measured sodium concentration reached 0 ppm. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 tert-butyl ester (1a) (properties: liquid).

[0086] (2) Synthesis of p-toluenesulfonyl PEG4 acid (2a) p-toluenesulfonyl PEG4 tert-butyl ester was placed in a 200 mL recovery flask, and 9.0 g of 36% aqueous HCl was added in excess of the p-toluenesulfonyl PEG4 tert-butyl ester. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. The reaction solution containing the product was dissolved in DCM and poured into a separatory funnel. Water was then added, the pH was measured using pH test paper, and separation was performed until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure and transferred to a 300 mL recovery flask. 100 mL of excess water was added, and the mixture was stirred at 60°C for 1 hour to decompose unreacted p-toluenesulfonyl chloride. The reaction solution was dissolved in DCM, placed in a separatory funnel, and water was added. The pH was measured using pH test paper, and separation was carried out until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 acid (2a) (state: liquid, yield: 39% based on the initial amount of raw materials charged).

[0087] (3) Synthesis of allylimidazolium p-toluenesulfonate (3b) 1.16 g of p-toluenesulfonyl PEG4 acid was placed in a 50 mL recovery flask, and 10 mL of toluene was added to dissolve the mixture. The recovery flask was then cooled on ice, and 0.36 g of 1-allylimidazole (1.1 equiv. relative to the p-toluenesulfonyl PEG4 acid) was added dropwise little by little. After the entire amount was added dropwise, the mixture was reacted at 80°C for 21 hours using a reflux condenser, resulting in layer separation into an upper layer containing unreacted material and a lower layer containing the product. The upper layer was discarded by decantation, and the lower layer containing the product was washed with diethyl ether (20 mL, 1 h, 3 times). After washing, it was dried under reduced pressure to obtain allylimidazolium p-toluenesulfonate (3b) (properties: liquid).

[0088] (4) Synthesis of allylimidazolium hydroxide (4b) and AimOE3C. Allylimidazolium p-toluenesulfonate was placed in a 110 mL vial and dissolved in 80 mL of methanol. Furthermore, 11 mL of strong anion exchange resin Amberlite IRN-78 (exchange capacity 1.1 eq. / L) was added and stirred at room temperature for 6 days to produce allylimidazolium hydroxide (4b). The reaction solution was concentrated into a 50 mL flask, and the neutralization reaction was allowed to proceed. Then, diethyl ether was added to the recovery flask containing the product and washed (20 mL, 30 min, 3 times). After washing, the mixture was dried under reduced pressure to obtain AimOE3C (properties: liquid).

[0089] AimOE3C: 1 H NMR (400MHz; DMSO; Me4Si) δ = 2.009 (3H, t, J = 7.0Hz, CH2COO), 3.390-3.548 (10H, m, NCH2CH2OCH2CH2OCH2CH2OCH2CH2COO), 3.797 and 4.380 (2H, t, J=5.0 and 4.8Hz, NCH2CH2OCH2), 4.915 (2H, d, NCH2CHCH2), 5.249-5.357 (2H, m, NCH2CHCH2), 6.016-6.114 (1H, m, NCH2CHCH2), 7.775 and 7.823 (1H, m, NCHCHN), 9.656 (1H, s, NCHN).

[0090] Synthesis Example 11 (VimOE3C(((R 1 = -CH = CH2, A = imidazolium cation, R 2 = CH2CH2, R 3 =CH2CH2, n=3, B=-COO -) Synthesis of Precursor—Vinylimidazolium p-toluenesulfonate) [Raw Materials] tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was purchased from Angene. p-toluenesulfonyl chloride, sodium hydroxide, and diethyl ether were purchased from Tokyo Chemical Industry Co., Ltd. 1-Vinylimidazole, toluene, dichloromethane, methanol, chloroform-d1, 99.8 atom% D with 0.03 vol% TMS, and dimethyl sulfoxide-d6, 99.9 atom% D with 0.03 vol% TMS were purchased from Kanto Chemical Co., Inc. Hydrochloric acid was purchased from FUJIFILM Wako Pure Chemical Corporation. Amberlite IRN-78, ion exchange resin, nuclear grade, was purchased from Alfa Aesar.

[0091] (1) Synthesis of p-toluenesulfonyl PEG4 tert-butyl ester (1a) 3.0 g of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate was placed in a 50 mL recovery flask and dissolved in 30 mL of DCM. Then, 1.6 g of NaOH (3.7 equiv. / molar equivalent) was added to the tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate. The recovery flask was then cooled on ice, and 1.3 equiv. / mol. of NaOH was added to the recovery flask. 2.6 g of p-toluenesulfonyl chloride was added dropwise little by little. After the entire amount was added dropwise, the mixture was stirred at room temperature for 24 hours to react, and solid impurities were removed using filter paper (5C, ADVANTEC). The reaction solution containing the filtered product was placed in a separatory funnel. Water was then added, and the sodium concentration of the aqueous layer was measured using a sodium concentration meter. Separation was continued until the measured sodium concentration reached 0 ppm. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 tert-butyl ester (1a) (properties: liquid).

[0092] (2) Synthesis of p-toluenesulfonyl PEG4 acid (2a) p-toluenesulfonyl PEG4 tert-butyl ester was placed in a 200 mL recovery flask, and 9.0 g of 36% aqueous HCl was added in excess of the p-toluenesulfonyl PEG4 tert-butyl ester. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. The reaction solution containing the product was dissolved in DCM and poured into a separatory funnel. Water was then added, the pH was measured using pH test paper, and separation was performed until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure and transferred to a 300 mL recovery flask. 100 mL of excess water was added, and the mixture was stirred at 60°C for 1 hour to decompose unreacted p-toluenesulfonyl chloride. The reaction solution was dissolved in DCM, placed in a separatory funnel, and water was added. The pH was measured using pH test paper, and separation was carried out until the pH of the aqueous layer reached 6. After separation, the DCM layer was dried under reduced pressure to obtain p-toluenesulfonyl PEG4 acid (2a) (state: liquid, yield: 39% based on the initial amount of raw materials charged).

[0093] (3) Synthesis of vinylimidazolium p-toluenesulfonate (3c) 0.5 g of p-toluenesulfonyl PEG4 acid was placed in a 50 mL recovery flask, and 10 mL of toluene was added to dissolve the mixture. The recovery flask was then cooled on ice, and 0.15 g of 1-vinylimidazole (1.2 equiv. relative to the p-toluenesulfonyl PEG4 acid) was added dropwise little by little. After the entire amount was added dropwise, the mixture was reacted at 80°C for 17 hours using a reflux condenser, resulting in layer separation into an upper layer containing unreacted material and a lower layer containing the product. The upper layer was discarded by decantation, and the lower layer containing the product was washed with diethyl ether (20 mL, 1 h, 3 times). After washing, the product was dried under reduced pressure to obtain vinylimidazolium p-toluenesulfonate (3b) (state: liquid).

[0094] Vinylimidazolium p-toluenesulfonate: 1H NMR (400MHz; DMSO; Me4Si) δ = 2.286 (3H, s, CHCCH3), 2.430 (2H, t, J = 6.4, CH2 COOH), 3.465-3.631 (10H, m, NCH2CH2OCH2CH2OCH2CH2OCH2CH2COOH), 3.798 and 4.379 (2H, t, J = both 5.0Hz, NCH2CH2OCH2), 5.431 (1H, dd, J = 2.8 and 2.4Hz, NCHCH2), 5.963 (1H, dd, J = 2.4Hz, NCHCH2), 7.121 and 7.490 (2H, d, J=both 7.6Hz, SO2CHCHCCH3CHCH), 7.274-7.350 (1H, m, NCHCH2), 7.880 and 8.199 (1H, m, NCHCHN), 9.408 (1H, s, NCHN), 12.181 (1H, s, CH2COOH).

[0095] (Cell Culture) Human melanoma fibroblasts (MAF1): MAF1 cells transfected with luciferase and / or fluorescent proteins (mEGFP or mCherry) were provided by Professor Erik Sahai (France-Click Institute). Mouse melanoma cells (5555): Same as above. Human kidney cells (BOSC): BOSC cells were provided by Professor Michiyuki Matsuda (Kyoto University). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere using a medium (high glucose containing L-glutamine and phenol red, Fujifilm Wako Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Co., Ltd.). The cells were divided into small portions using a trypsin solution (0.5 w / v% trypsin-5.3 mmol / L EDTA·4Na solution, phenol red-free, Fujifilm Wako Co., Ltd.).

[0096] (Single cell preservation experiment and the percentage of surviving cells before and after preservation) 1.0 x 10 6Cells were collected in a 1.5 mL tube and centrifuged (100 g, 5 minutes at room temperature). After removing the supernatant, 100 μL of CPA solution C (a mixed solution of zwitterion, DMSO, EG, and water) was added and pipetted. The resulting samples were immediately stored in a refrigerator at -25°C for 3 to 30 days. The samples were thawed with 1 mL of medium at 37°C and centrifuged (100 g, 5 minutes at room temperature). After removing the supernatant, cell viability was measured using trypan blue (Fujifilm Wako Pure Chemical Industries, Ltd.) and a hemocytometer (Fukae Kasei Co., Ltd., Watson). Generally, viability is defined as the percentage of viable cells among all cells in a given cell population. However, counting dead cells is difficult, and repeated observation of the same cell population often results in significant variability. Therefore, the effectiveness of refrigerated storage was evaluated herein by the number of viable cells before and after storage. This value is sometimes referred to as cell recovery rate or simply recovery rate. Proportion of viable cells before and after storage = Cell recovery rate (%) = (number of viable cells after storage (sample)) / (number of cells before storage (sample)) × 100

[0097] (Culture toxicity to single cells) 1.0 x 10 6 The cells were collected in a 1.5 mL tube and centrifuged (100 G, room temperature for 5 minutes). After removing the supernatant, 100 μL of CPA solution was added and pipetted. After incubation at 37°C for 1 hour, cell viability was counted using a hemocytometer and trypan blue.

[0098] (Spheroids) Spheroids were prepared by the hanging drop method. 2.0 × 10 cells were cultured to obtain spheroids. 6 To each cell, medium and 1 mL of 1.2 w / v% methylcellulose (Sigma-Aldrich) / DMEM (Fujifilm Wako Co., Ltd.) were added to make a 2 mL solution, and 20 μL of this solution was dropped onto the underside of the culture plate lid. The culture plate was then filled with PBS (Fujifilm Wako Co., Ltd.), and the culture plate lid was closed. The cells were cultured statically in a humidified incubator at 37°C in a 5% CO atmosphere to obtain spheroids.

[0099] (Spheroid preservation experiment and measurement of viability before and after preservation) Four spheroids were collected in a 1.5 mL tube and washed with 1 mL of PBS. After removing the supernatant, 100 μL of test solution was added. The samples were stored in a -25°C refrigerator for 3 to 30 days. After storage, 1 mL of 37°C medium was added, and the supernatant was removed. The samples were washed with 1 mL of PBS for 3 minutes, and the supernatant was removed. 50 μL of trypsin solution was added and incubated at 37°C for 30 minutes. 50 μL of medium was added, and the spheroids were disrupted by pipetting. 10 μL of cell suspension was added to a 1.5 mL tube and mixed with 10 μL of trypan blue (Fujifilm Wako). The viability of spheroid-derived cells was counted using a hemocytometer and calculated using the following formula. The cell recovery rate of the spheroids was calculated from the number of surviving cells that constituted the spheroids before and after preservation. This may be simply referred to as cell recovery rate or recovery rate. The percentage of viable cells that make up the spheroid before and after storage = Spheroid cell recovery rate (%) = (number of viable cells derived from the spheroid after storage (sample)) / (number of cells derived from the spheroid before storage (sample)) × 100

[0100] The recovered spheroids were cultured for 24 hours in a humidified incubator at 37°C in a 5% CO2 atmosphere. The culture medium used was a high glucose medium containing L-glutamine and phenol red (Fujifilm Wako Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Co., Ltd.). The cell recovery rate of the spheroids after 24 hours was calculated using the following formula. The cell count was evaluated by disrupting the cells with trypsin in the same manner as above. Cell recovery rate (%) = (number of viable cells after 24 hours of culture (sample)) / (number of viable cells after 24 hours of culture (unfrozen)) x 100

[0101] (Spheroid Toxicity) Four spheroids were collected in a 1.5 mL tube and washed with 1 mL of PBS. After removing the supernatant, 100 μL of solution was added. The cell recovery rate of the spheroids was then counted using a hemocytometer and trypan blue.

[0102] (DSC) The melting point and freezing point of the solution were measured using a differential scanning calorimeter (DSC60-A; Shimadzu Corporation). The solution (10 μL) was added to an aluminum pan and placed in the sample chamber of the calorimeter. The sample was cooled to -100°C at 1°C / min and heated to 25°C at 5°C / min.

[0103] (Confirmation of Supercooling) Whether supercooling occurred was determined from the results of DSC.

[0104] (FT-IR) Infrared (IR) spectra were measured using a Nicolet 10 (Thermo Fisher Scientific).

[0105] (Example 1) (Evaluation of Physical Properties of OE2imC3C / DMSO / EG) A composition was prepared as follows: OE2imC3C 10 parts by mass, DMSO 5 parts by mass, EG 5 parts by mass, water 80 parts by mass, total 100 parts by mass, and the ratio of DMSO to EG was 1: 1. Specifically, the mass ratios of OE2imC3C / DMSO / EG were 10 / 5 / 5, 10 / 10 / 10, 10 / 15 / 15, 10 / 20 / 20, and 10 / 25 / 25, and the compositions are shown in Table 1.

[0106] (Tm, Tf, frozen state, and supercooling results) Tm and Tf were measured by DSC. The DSC chart is shown in FIG. 1, and the freezing point (Tf / °C) and melting point (Tm / °C) are shown in Table 1. Table 1 also summarizes whether freezing occurred at the storage temperature (-25°C) (◯: not frozen, ×: frozen, △: varied depending on the lot) and whether supercooling occurred.

[0107]

[0108] (Effect of Non-Cryopreservation) MAF1 spheroids were stored at -25°C for 3 days with different composition ratios, and the cell recovery and viability after storage were examined. The results are shown in Table 1 and Figure 2. Good results were obtained with compositions that did not have a Tm at the storage temperature, were not visually observed to be frozen, and were not supercooled. Note that the viability in this case is expressed as (number of living cells in the cell group to be evaluated) / (number of living cells in the cell group to be evaluated + number of dead cells in the cell group to be evaluated) × 100% for the sample to be evaluated.

[0109] Example 2: Physicochemical Properties and Biological Evaluation of C1imC3C / DMSO / EG A composition was prepared using C1imC3C instead of OE2imC3C in Example 1, and MAF1 spheroids were stored at -25°C for 20 days. Physicochemical properties and biological evaluation were performed after storage. The results are shown in Table 1 and Figure 3. Although no Example number is given in Table 1, the results of a similar experiment using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20) are also shown in Table 1 and Figure 3. Example 3: Evaluation of Physical Properties of C4imC3S / DMSO / EG MAF1 spheroids were stored at -25°C for 20 days using a composition of C4imC3S (20 / 20 / 20) instead of C1imC3C in Example 2. The recovery rate and viability are shown in Table 1 and Figure 3. The viability was calculated using the same method as in Example 2.

[0110] (Reference Examples 1 and 2) Evaluation of Physical Properties of OE2imC3C / DMSO (Containing Only One Additive (X)) (1) Regarding the recovery rate of MAF1 in a composition consisting of OE2imC3C / DMSO after storage at −25°C for 3 days, Table 2 and Figure 4 show that the recovery rate deteriorates when the composition becomes solid (S) at the storage temperature. (2) Compositions consisting of OE2imC3C / DMSO were prepared at 10 / 10, 10 / 20, 10 / 30, 10 / 40, and 10 / 50, and the recovery rates of MAF1 spheroids after storage at −25°C for 3 days are shown in Figure 5. The zwitterion to additive ratio was 10 / 40, which is the same as the 10 / 20 / 20 ratio used as the standard in Examples 2 and 3, but the viability and recovery rate were poor. One reason for this is thought to be that the addition of EG relatively reduced the concentration of DMSO.

[0111]

[0112] Example 4 Table 3 and FIG. 6 show the change in cell recovery rate when MAF1 spheroids were stored at −25° C. for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20).

[0113] Example 5 The change in cell recovery rate when 5555 spheroids were stored at −25° C. for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20) is shown in Table 3 and FIG.

[0114] (Example 6) Table 3 and Figure 8 show the change in cell recovery rate when BOSC spheroids were stored at -25°C for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20).

[0115]

[0116] Example 7 Table 4 and FIG. 9 show the change in cell recovery rate when MAF1 cells were stored at −25° C. for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20).

[0117] (Example 8) Table 4 and Figure 10 show the change in cell recovery rate when 5555 cells were stored at -25°C for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20).

[0118] (Example 9) The change in cell recovery rate when BOSC cells were stored at -25°C for 30 days using a composition of OE2imC3C / DMSO / EG (10 / 20 / 20) is shown in Table 4 and Figure 11 .

[0119]

[0120] Reference Example 3 FIG. 12 shows the change in recovery rate over time when MAF1 spheroids were stored at −25° C. for 30 days using a composition that used 0.9% NaCl instead of the liquid zwitterion.

[0121] Reference Example 4 When MAF1 spheroids were stored at −25° C. for 30 days using a composition that used sucrose instead of the zwitterionic liquid, the recovery rate was measured. However, the spheroids were frozen and the recovery rate was low.

[0122] Reference Example 5 Figure 13 shows an optical microscope photograph (right in Figure 13) of MDA-MB-231 cells stored at -80°C in a 10% by mass OE2imC3C aqueous solution, and an optical microscope photograph (left in Figure 13) of MAF1 cell spheroids stored in culture medium at room temperature. As can be seen from the left in Figure 13, spheroids are three-dimensional cell masses with diameters of approximately 10 to several hundred μm, and if ridges develop during cryopreservation, the spheroids will be damaged and their preservation status will deteriorate. Also, from the right in Figure 13, the ridges visible at 50 μm intervals are ice crystals, and during single-cell preservation, cells can exist between these ridges, making cryopreservation possible.

[0123] Example 10: MAF1 spheroids were stored at -25°C for 10 days in test solutions containing DMSO (20 wt%), EG (20 wt%), and various ratios of the zwitterionic compounds OE2imC3C, OE2imC3S, C1imC3C, and C1imC3S. The differences in cell recovery rates when the ratios of the zwitterionic compounds were varied were investigated. The results are shown in Table 5 and Figure 14.

[0124]

[0125] Example 11: MAF1 spheroids were treated with test solutions containing DMSO (20 wt%), EG (20 wt%), and various ratios of the zwitterionic compounds OE2imC3C, OE2imC3S, C1imC3C, and C1imC3S for 1 hour at 0°C to examine the difference in cell recovery rate when the ratio of each zwitterionic compound was changed. The results are shown in Table 6 and Figure 15.

[0126]

[0127] Example 12: The recovery rate of MAF1 spheroids was investigated when they were stored at −85°C for 30 days using test solutions containing different compositions in which the ratios of the zwitterionic compounds OE2imC3C, OE2imC3S, C1imC3C, and C1imC3S were varied in addition to DMSO (20 wt%) and EG (20 wt%). As a result, it was confirmed that there are combinations of the compositions of the present invention that do not freeze even at −85°C.

[0128] By using the non-cryopreservation composition of the present invention, the multicellular population can be preserved in an unfrozen state because it does not have a melting point at storage temperatures, does not vitrify, does not supercool, and does not freeze even at temperatures below −25° C. Therefore, regenerative medicine using the multicellular population becomes industrially feasible.

Claims

1. A composition for non-cryopreservation of a multicellular population, comprising one or more aprotic zwitterionic compounds (A) and two or more additives (X) compatible with component (A).

2. The aprotic zwitterionic compound (A) is represented by the general formula (1) (R 1 represents a linear alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, or a linear C alkyl-(OCHCH)-; A represents a cation portion of a zwitterion and is selected from an imidazolium cation, a phosphonium cation, an ammonium cation, a sulfonium cation, a pyrazolium cation, a pyridinium cation, a pyrrolidinium cation, a morpholinium cation, a cyclopropenium cation, and a piperidinium cation; R 2 represents an alkylene group having 1 to 4 carbon atoms; R 3 2. The non-cryopreservation composition according to claim 1, which is an aprotic zwitterionic compound represented by the following formula: wherein R represents an alkylene group having 2 to 4 carbon atoms; m represents a number of 1 or 2; n represents a number of 0 to 10; B represents an anion selected from -SO3-, -COO-, -OP=O(H)O-, -OP=O(CH3)O-, -OP=O(OR4)O-, and -OP=O(OH)O-; and R4 represents an alkyl group having 1 to 8 carbon atoms which may have a heteroatom.

3. The non-cryopreservation composition according to claim 1, wherein the additive X is an additive selected from the group consisting of: (i) a small molecule compound having no hydroxyl group or a compound having planarity, (ii) a compound having one or more hydroxyl groups, (iii) a peptide or a protein, (iv) a polymer compound, (v) a surfactant, and (vi) a zwitterionic polymer or zwitterionic oligomer represented by the following general formula (2): (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, and are each represented by -COO-, -SO3-, -OP=O(H)O-, -OP=O(CH3)O-, and -OP=O(OR 5 ) O-, where R 5 represents a hydrogen atom or a methyl group; Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a straight-chain or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m and n each represent a number indicating the content ratio of each repeating unit and satisfying 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s and t each represent an integer of 0 to 6.

4. A method for preserving a multicellular population under non-freezing conditions, comprising using the composition according to any one of claims 1 to 3.