Cryoprotectant for cells

Abstract

The present invention provides a cryoprotectant for cells that contains an amphoteric polymer having a specific cationic group and a specific anionic group, a hydrophobic amino acid, and a water-soluble cellulose.

Description

Cryoprotectants for cells

[0001] The present invention relates to a cryoprotectant capable of cryopreserving cells and reducing damage to cells after thawing.

[0002] Because cells continually change over time and depending on the surrounding environment, they must be appropriately preserved to maintain their properties. Currently, this is often achieved through cryopreservation. Traditionally, cells have typically been cryopreserved in a dispersed state. However, for actual use, cells must be cultured in two or three dimensions to achieve appropriate cell function and morphology. In recent years, numerous two-dimensional and three-dimensional culture methods have been developed that incorporate complex structures or multiple cell types to more closely mimic the in vivo environment, resulting in longer culture periods. Therefore, starting with cell culture each time cells are used requires a long time before they can actually be used. Furthermore, as mentioned above, two-dimensional and three-dimensional culture methods that incorporate complex structures or multiple cell types require skilled culture techniques, hindering the widespread adoption of cell culture techniques. Furthermore, starting with cell culture each time cells are used results inconsistencies due to differences in the culture process, which is undesirable from the perspective of obtaining consistent measurement results. Therefore, in addition to cell viability after cryopreservation and thawing, maintaining cell function and morphology after cryopreservation and thawing is also essential.

[0003] Given the above-mentioned circumstances, there is a need for cryoprotectants that can cryopreserve 2D or 3D cultured cells while maintaining their morphology and function. However, when common cryoprotectants for dispersed cells, such as dimethyl sulfoxide (DMSO), are applied to 2D or 3D cultured cells, most cells die after cryopreservation and thawing. Therefore, efforts have been made to develop new cryoprotectants that can be used on 2D or 3D cultured cells. Among these, the development of cryoprotectants using amphoteric polymers has been active in recent years.

[0004] For example, Patent Document 1 reports that an amphoteric polymer represented by the following formula (8) has a cryoprotective effect on two-dimensionally cultured cells, and that the use of this amphoteric polymer during cryopreservation of cells results in a high cell survival rate after thawing.

[0005]

[0006] Furthermore, Non-Patent Document 1 reports that a carboxylated polylysine (PLL-GA) represented by the following formula has a cryoprotective effect on two-dimensionally or three-dimensionally cultured cells, and that the use of this carboxylated polylysine during cryopreservation of cells results in a high cell survival rate after thawing.

[0007]

[0008] Amphoteric polymers are known to have the effect of inhibiting ice crystal formation, and the use of amphoteric polymers during cryopreservation of cells inhibits the formation of ice crystals inside and outside the cells, thereby increasing the cell survival rate after thawing. However, the use of amphoteric polymers alone poses the problem of making it difficult to maintain the function and morphology of cells after thawing.

[0009] Furthermore, Non-Patent Document 2 reports that proline has the effect of alleviating osmotic stress on cells during freezing, and that its cryoprotective effect on two-dimensionally cultured cells results in high cell survival rates after thawing. However, there is a problem in that the use of proline alone during cell cryopreservation makes it difficult to maintain the function and morphology of cells after thawing.

[0010] International Publication No. 2019 / 175596

[0011] Biomacromolecules 2020, 21, 3017-3025Cryobiology 71 (2015) 472-480

[0012] As described above, attempts have been made to develop cryoprotectants for cells cultured in two or three dimensions, and some have been shown to be effective in improving cell viability after thawing; however, no cryoprotectant capable of maintaining cell morphology or function has yet been developed.

[0013] An object of the present invention is to provide a cryoprotectant that can maintain not only cell viability but also cell morphology and function after cryopreservation and thawing of cells (preferably cells cultured in two or three dimensions).

[0014] As a result of extensive research aimed at achieving the above-mentioned object, the present inventors have found that the above-mentioned problems can be solved by using a cryoprotectant for cells (hereinafter sometimes abbreviated as "the cryoprotectant of the present invention") that contains an amphoteric polymer having a specific cationic group and a specific anionic group, a hydrophobic amino acid, and water-soluble cellulose. Based on this finding, the present invention is as follows.

[0015] [1] A cryoprotectant for cells comprising an amphoteric polymer, a hydrophobic amino acid, and water-soluble cellulose, wherein the amphoteric polymer is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , *-N ＋ (CH ３ ) ２ -*, *-S ＋ (CH ３ ) ２ , and *-S ＋ (CH ３ a cationic group selected from the group consisting of *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-O-P(O)(O － and an anionic group selected from the group consisting of: -O-* (wherein * represents a bonding position).

[0016] [2] The amphoteric polymer is represented by the formula (1):

[0017]

[0018] (In formula (1), * represents a bonding position, R １ and R ２ each independently represents a hydrogen atom or a methyl group; ３ is absent or represents a methylene group;４ O represents an oxyalkylene group having 2 to 4 carbon atoms, and n represents a number from 0 to 100.) and a repeating structure A represented by formula (2):

[0019]

[0020] (In formula (2), * represents a bonding position, R ３ represents a hydrogen atom or a methyl group; １ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents the bonding position), p represents an integer of 1 to 6, and X １ is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position) and a repeating structure B represented by formula (3):

[0021]

[0022] (In formula (3), * represents a bonding position, R ４ represents a hydrogen atom or a methyl group; ２ is absent or represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), q represents an integer of 0 to 6, and Y １ is *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S(O) ２ (O － ) (wherein * represents a bonding position, and C ６ H４ represents a phenylene group.) represents a group selected from the group consisting of: (a) a repeating structure C represented by the following formula: and (b) a repeating structure B represented by the following formula: and (c) a molar ratio of the repeating structure B to the sum of the repeating structure B and the repeating structure C is 0.1 or more and 0.9 or less.

[0023] [3] In the formula (1), R １ is a hydrogen atom, and R ２ is a methyl group, and A ３ is absent or is a methylene group, and A ４ O is an oxyethylene group, and n is a number from 0 to 50, and in the formula (2), R ３ is a hydrogen atom, and A １ is a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), p is an integer of 2 to 4, and X １ *-N ＋ (CH ３ ) ２ H and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position), and in formula (3), R ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － (wherein * represents a bonding position.) [4] The cryoprotectant according to [3], wherein p is 2.

[0024] [5] The amphoteric polymer is represented by the formula (4):

[0025]

[0026] (In formula (4), * represents a bonding position, R ５ represents a hydrogen atom or a methyl group; ５ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), r represents an integer of 1 to 6, and X ２ is *-N＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position)) and a repeating structure D represented by formula (5):

[0027]

[0028] (In formula (5), * represents a bonding position, R ６ represents a hydrogen atom or a methyl group; ６ is absent or represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), s represents an integer of 0 to 6, and Y ２ is *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S(O) ２ (O － ) (wherein * represents a bonding position, and C ６ H ４ represents a phenylene group.) represents a group selected from the group consisting of: (a) a repeating structure E represented by: and (b) a repeating structure D represented by: and (c) a molar ratio of the repeating structure D to the sum of the repeating structure D and the repeating structure E is 0.1 or more and 0.9 or less.

[0029] [6] In the formula (4), R ５ is a methyl group, and A ５ is *-CO-O-* (wherein * represents a bonding position), r is 2, and X ２ *-N ＋ (CH ３ )２ H (in the formula, * represents a bonding position), and ６ is a methyl group, and A ６ is absent or is *-CO-O-* (wherein * represents a bonding position), s is an integer of 0 to 3, and Y ２ *-CO-O － or *-S(O) ２ (O － ) (wherein * represents a binding position).

[0030] [7] The amphoteric polymer is represented by the formula (6):

[0031]

[0032] (In formula (6), * represents a bonding position), or a group represented by formula (7):

[0033]

[0034] (In formula (7), * represents a bonding position.) The cryoprotectant according to [1] above, having a group represented by the following formula:

[0035] [8] The cryoprotectant according to any one of [1] to [7], wherein the number-average molecular weight of the amphoteric polymer is 1,000 to 2,000,000. [9] The cryoprotectant according to any one of [1] to [8], wherein the hydrophobic amino acid is proline.

[10] The cryoprotectant according to any one of [1] to [9], wherein the water-soluble cellulose is at least one selected from the group consisting of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose.

[0036]

[11] A method for cryopreserving cells, comprising: Step 1: mixing cells with the cryoprotectant according to any one of [1] to

[10] ; and Step 2: freezing the mixture containing the cells and the cryoprotectant.

[0037]

[12] The method according to

[11] above, wherein the cells are two-dimensionally cultured cells, and the method comprises, prior to step 1, step A of two-dimensionally culturing adherent cells in a medium on a culture substrate, step 1 being step C of mixing the two-dimensionally cultured cells with the cryoprotectant according to any one of [1] to

[10] above, and step 2 being step D of freezing the mixture containing the two-dimensionally cultured cells on the culture substrate and the cryoprotectant.

[13] The method according to

[12] above, comprising step B of removing a culture supernatant from the two-dimensionally cultured cells between steps A and C.

[0038]

[14] The method according to

[11] above, wherein the cells are three-dimensionally cultured cells, and the method comprises, prior to step 1, step E of three-dimensionally culturing the cells in a medium, wherein step 1 is step G of mixing the three-dimensionally cultured cells with the cryoprotectant according to any one of [1] to

[10] above, and step 2 is step H of freezing the mixture containing the three-dimensionally cultured cells and the cryoprotectant.

[15] The method according to

[14] above, comprising, between step E and step G, step F of transferring the three-dimensionally cultured cells to a cryopreservation container and removing the culture supernatant.

[0039]

[16] The method according to

[11] above, wherein the method for cryopreserving cells is a method for cryopreserving cells suspended in a medium, and comprises, prior to step 1, step I of suspending the cells in a medium, wherein step 1 is step K, and step 2 is step L.

[17] The method according to

[16] above, comprising, between step I and step K, step J of removing a supernatant from a mixture containing the medium and the cells.

[0040] By using the cryoprotectant of the present invention, not only the cell viability but also the morphology and function of cells can be maintained after cryopreservation and thawing of cells.

[0041] The present invention will be described below in order. The descriptions in this specification can be combined with each other unless it is clear that they cannot be combined.

[0042] As used herein, the term "cryoprotectant for cells" refers to an additive that can reduce damage to cells during cryopreservation and after thawing.

[0043] The cryoprotectant of the present invention comprises an amphoteric polymer having a specific cationic group and a specific anionic group, a hydrophobic amino acid, and water-soluble cellulose. Furthermore, the cryoprotectant of the present invention may contain water, a medium, and other components to the extent that the effects of the present invention are not impaired. The following describes the cells and each component contained in the cryoprotectant of the present invention.

[0044] [Cells] Examples of cells include established cells for culture, fertilized eggs and egg cells of animals including humans. Examples of cells include stem cells such as sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and umbilical cord blood cells, as well as animal cells including humans such as liver cells, nerve cells, cardiac muscle cells, vascular endothelial cells, vascular smooth muscle cells, and blood cells. Examples of cells include plant cells.

[0045] As used herein, "two-dimensionally culturing cells" refers to culturing cells in a monolayer on a culture substrate, and "three-dimensionally culturing cells" refers to culturing cells with a vertical thickness. As used herein, "two-dimensionally cultured cells" refers to cells obtained by two-dimensional culture, and "three-dimensionally cultured cells" refers to cells obtained by three-dimensional culture.

[0046] The cryoprotective agent of the present invention is preferably used to protect two-dimensionally cultured cells, three-dimensionally cultured cells, or cells suspended in a medium, more preferably two-dimensionally cultured cells or three-dimensionally cultured cells. Therefore, the cryoprotective agent of the present invention is preferably used for two-dimensionally cultured cells, three-dimensionally cultured cells, or cells suspended in a medium, more preferably two-dimensionally cultured cells or three-dimensionally cultured cells.

[0047] Examples of two-dimensionally cultured cells include cells adhered to a culture substrate and monolayer cell membranes formed by cells adhering to each other. Examples of three-dimensionally cultured cells include spheroids and organoids formed by cells aggregating and adhering to each other, and three-dimensionally cultured cell sheets and biological tissues formed by cells stacking on each other. The constituent cells of either two-dimensional or three-dimensionally cultured cells are not limited to one type, and may be formed by mixing two or more types of cells.

[0048] The amount of the cryoprotectant used in the present invention is 1×10 ４ The amount of solution is preferably 1 to 2,000 μL, more preferably 2 to 1,500 μL, and even more preferably 5 to 1,000 μL per cell.

[0049] The amount of the cryoprotectant used in the present invention is 1×10 ４ The amount of solution is preferably 10 to 300 μL, more preferably 20 to 200 μL, even more preferably 50 to 200 μL, and particularly preferably 50 to 100 μL per unit of two-dimensionally cultured cells.

[0050] The amount of the cryoprotectant used in the present invention is 1×10 ４ The amount of solution is preferably 100 to 2,000 μL, more preferably 200 to 1,500 μL, and even more preferably 500 to 1,000 μL per 3D cultured cell.

[0051] The amount of the cryoprotectant used in the present invention is 1×10 ４ The amount is preferably 1 to 20 μL, more preferably 2 to 15 μL, and even more preferably 5 to 10 μL per cell suspended in the medium.

[0052] [Amphoteric Polymer] The amphoteric polymer used in the present invention may be either a polyampholite or a zwitterionic polymer. In this specification, "polyampholite" refers to a polymer in which cationic and anionic groups are not present in the same side chain (e.g., a polymer having anionic groups as pendant groups and cationic groups in the side chain), and "zwitterionic polymer" refers to a polymer in which cationic and anionic groups are present in the same side chain.

[0053] The amphoteric polymer used in the present invention is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , *-N ＋ (CH ３ ) ２ -*, *-S ＋ (CH ３ ) ２ , and *-S ＋ (CH ３ a cationic group selected from the group consisting of *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-O-P(O)(O － )-O-* (where * represents a bonding position). Here, "*" in the above formula or other formulas represents a bonding position, not a carbon atom. Therefore, "*-" in this specification represents a single bond.

[0054] In this specification, groups represented by formulas using element symbols, parentheses, "-" and "*" have the same meaning as commonly understood by a person skilled in the chemical arts. For example, "*-N ＋ (CH ３ ) H ２ " is N ＋ One CH ３and a group in which two H are bonded (i.e., a methylammonio group), and "*-S(O) ２ (O － ) is a combination of two O and one O in S. － is bonded (i.e., a sulfo group from which a proton has been removed), and － "-P(O)(OH)(O)" － )) is a group consisting of P, O, OH and O － is bonded (i.e., "*-O-P(O)(OH)(O － ) refers to a phosphate group with one proton removed.

[0055] <Polyampholite (Amphoteric Polymer 1)> A preferred amphoteric polymer in one embodiment of the present invention is represented by the formula (1):

[0056]

[0057] (In formula (1), * represents a bonding position, R １ and R ２ each independently represents a hydrogen atom or a methyl group; ３ is absent or represents a methylene group; ４ O represents an oxyalkylene group having 2 to 4 carbon atoms, and n represents a number from 1 to 100.) and a repeating structure A represented by formula (2):

[0058]

[0059] (In formula (2), * represents a bonding position, R ３ represents a hydrogen atom or a methyl group; １ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents the bonding position), p represents an integer of 1 to 6, and X １ is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ )３ , and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position) and a repeating structure B represented by formula (3):

[0060]

[0061] (In formula (3), * represents a bonding position, R ４ represents a hydrogen atom or a methyl group; ２ is absent or represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), q represents an integer of 0 to 6, and Y １ is *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S(O) ２ (O － ) (wherein * represents a bonding position, and C ６ H ４ represents a phenylene group.) represents a group selected from the group consisting of: and a polyampholite (sometimes referred to as "amphoteric polymer 1" in this specification) having a repeating structure C represented by:

[0062] A in formula (2) １ *-CO-O-* and *-CO-NH-* are both A in formula (2). １ A in formula (2) is bonded to the carbon atom (C) above at the "*-CO" side. １ From the viewpoint of the stability of the amphoteric polymer 1, is preferably *--CO--NH--*.

[0063] A in formula (3) ２ *-CO-O-* and *-CO-NH-* are both A in formula (3). ２ It is bonded to the carbon atom (C) above at the "*-CO" side.

[0064] In this specification, the term "repeating structure" refers to a partial structure that appears repeatedly in a polymer. This repeating structure may be a repeating unit itself or a part of a repeating unit.

[0065] In this specification, the term "repeating unit" refers to a partial structure formed from a monomer that appears repeatedly in a polymer. Therefore, when a polymer is formed by polymerization of multiple types of monomers, the ratio of each monomer to all monomers is the same as the ratio of repeating units formed from that monomer to all repeating units.

[0066] In this specification, "A ３ "does not exist" means that A in formula (1) ３ This means that the carbon atom (C) above and the oxygen atom (O) below it are bonded by a single bond. ３ Other expressions similar to "does not exist" have the same meaning.

[0067] In this specification, "n is 0" means that (A ４ O) ｎ does not exist, and (A ４ O) ｎ The oxygen atom (O) above and the R below ２ and are bonded by a single bond. Other expressions similar to "n is 0" have the same meaning. Also, n in formula (1) is an oxyalkylene group (A ４ 0), and is in fact an average value. Therefore, n may be a decimal number.

[0068] Examples of the repeating structure A represented by the formula (1) include the following: １ is a hydrogen atom, and R ２ is a methyl group, and A ３ A repeating structure A in which R is absent and n is 0, and １ is a hydrogen atom, and R ２ is a methyl group, and A ３ is a methylene group, and A ４A repeating structure A in which O represents an oxyalkylene group having 2 to 4 carbon atoms (preferably 2 carbon atoms), and n is a number of 5 to 50 (preferably 5 to 45).

[0069] In view of the solubility of the amphoteric polymer 1 in water, preferred examples of the repeating structure A include the following: １ is a hydrogen atom, and R ２ is a methyl group, and A ３ A repeating structure A in which R is absent and n is 0, and １ is a hydrogen atom, and R ２ is a methyl group, and A ３ is a methylene group, and A ４ A repeating structure A in which O is an oxyalkylene group having 2 or 3 carbon atoms (preferably 2 carbon atoms), and n is a number from 5 to 50 (preferably 5 to 45).

[0070] From the viewpoint of the solubility of the amphoteric polymer in water, more preferred examples of the repeating structure A include the following: １ is a hydrogen atom, and R ２ is a methyl group, and A ３ A repeating structure A in which R is absent and n is 0, and １ is a hydrogen atom, and R ２ is a methyl group, and A ３ is a methylene group, and A ４ A repeating structure A in which O is an oxyethylene group and n is a number from 5 to 50 (preferably 5 to 45).

[0071] Examples of the repeating structure B represented by the formula (2) include the following (in the formula, * represents a bonding position): ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-N ＋ (CH ３ ) ２ Repeating structures B, R ３ is a hydrogen atom, and A １is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-N ＋ (CH ３ ) ２ Repeating structures B, R ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-S ＋ (CH ３ ) ２ The repeating structure B, R ３ is a hydrogen atom, and A １ is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-S ＋ (CH ３ ) ２ The repeating structure B, R ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X is *-N ＋ H ３ A repeating structure B, ３ is a hydrogen atom, and A １ is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X is *-N ＋ H ３ Repeating structure B.

[0072] From the viewpoint of the solubility of the amphoteric polymer 1 in water, preferred examples of the repeating structure B include the following (in the following formula, * indicates a bonding position): ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-N ＋ (CH ３ ) ２ Repeating structures B, R ３ is a hydrogen atom, and A １ is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X１ *-N ＋ (CH ３ ) ２ Repeating structures B, R ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-S ＋ (CH ３ ) ２ A repeating structure B, ３ is a hydrogen atom, and A １ is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-S ＋ (CH ３ ) ２ Repeating structure B.

[0073] From the viewpoint of the solubility of the amphoteric polymer 1 in water, more preferable examples of the repeating structure B include the following (in the following formula, * represents a bonding position): ３ is a hydrogen atom, and A １ is *-CO-NH-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-N ＋ (CH ３ ) ２ H, and R ３ is a hydrogen atom, and A １ is *-CO-O-*, p is an integer of 2 to 4 (preferably p is 2), and X １ *-S ＋ (CH ３ ) ２ Repeating structure B.

[0074] Examples of the repeating structure C represented by the formula (3) include the following (in the formula, * represents a bonding position): ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － A repeating structure C, R ４ is a hydrogen atom, and A２ is *-CO-O-*, q is 2, and Y １ *-CO-O － A repeating structure C, R ４ is a hydrogen atom, and A ２ is *-CO-O-*, q is 2, and Y １ *-S(O) ２ (O － ) repeating structure C, R ４ is a hydrogen atom, and A ２ is *-CO-O-*, q is 2, and Y １ is *-OP(O)(OH)(O － ), and R ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-C ６ H ４ -S(O) ２ (O － ) repeating structure C.

[0075] From the viewpoint of ease of synthesis of the amphoteric polymer 1, preferred examples of the repeating structure C include the following (in the following formula, * represents a bonding position, and C ６ H ４ represents a phenylene group): R ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － A repeating structure C, R ４ is a hydrogen atom, and A ２ is *-CO-O-*, q is 2, and Y １ *-CO-O － a repeating structure C, and ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-C ６ H ４ -S(O) ２ (O － ) repeating structure C.

[0076] From the viewpoint of ease of synthesis of the amphoteric polymer 1, more preferred examples of the repeating structure C include the following (in the following formula, * represents a bonding position): ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － a repeating structure C, and ４ is a hydrogen atom, and A ２ is *-CO-O-*, q is 2, and Y １ *-CO-O － A repeating structure C.

[0077] From the viewpoint of ease of synthesis of the amphoteric polymer 1, more preferred examples of the repeating structure C include the following (in the following formula, * represents a bonding position): ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － A repeating structure C.

[0078] In view of the cell cryoprotective effect of the amphoteric polymer 1, in the formula (1), R １ is a hydrogen atom, and R ２ is a methyl group, and A ３ is absent or is a methylene group, and A ４ O is an oxyethylene group, and n is a number of 0 to 50 (more preferably 5 to 50, and even more preferably 5 to 45), and in the formula (2), R ３ is a hydrogen atom, and A １ is a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position) (more preferably *-CO-NH-*), p is an integer of 2 to 4 (preferably p is 2), and X １ *-N ＋ (CH ３ ) ２ H and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position), and in formula (3), R４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － (wherein * represents a bonding position) is preferable.

[0079] From the viewpoint of the cryoprotective effect and cytotoxicity of amphoteric polymer 1, the molar ratio of the repeating structure B to the sum of the repeating structures B and C (i.e., the amount (moles) of the repeating structure B / (the amount (moles) of the repeating structure B+the amount (moles) of the repeating structure C)) is preferably 0.1 or more and 0.9 or less. If the molar ratio is less than 0.1, the amphoteric polymer 1 becomes strongly anionic and is less likely to remain around cells, which may result in the cryoprotective effect not being exerted. If the molar ratio exceeds 0.9, the amphoteric polymer 1 becomes strongly cationic and may exhibit cytotoxicity. From the viewpoint of the cryoprotective effect and cytotoxicity of amphoteric polymer 1, the molar ratio is more preferably 0.2 or more and 0.7 or less, and even more preferably 0.2 or more and 0.5 or less.

[0080] From the viewpoint of the cryoprotective effect of the amphoteric polymer 1, the proportion of the repeating structure A in the amphoteric polymer 1 is preferably 70 mol % or less, and more preferably 60 mol % or less, based on the total repeating units of the amphoteric polymer 1. The repeating structure A is usually a repeating unit.

[0081] From the viewpoint of the solubility of the amphoteric polymer 1 in water, the proportion of the repeating structure A in the amphoteric polymer 1 is preferably 25 mol % or more, and more preferably 35 mol % or more, based on the total repeating units of the amphoteric polymer 1.

[0082] In the amphoteric polymer 1, the repeating unit B and the repeating unit C are preferably bonded together. That is, the amphoteric polymer 1 is represented by the formula (2-3):

[0083]

[0084] (The definitions of the symbols in formula (2-3) are as defined in formula (2) and formula (3).) It is preferable that the repeating structure B-C is represented by the following formula: The repeating structure B-C is usually a repeating unit.

[0085] In the amphoteric polymer 1, two repeating units C may be bonded together. That is, the amphoteric polymer 1 is represented by the formula (3-3):

[0086]

[0087] (The definitions of the symbols in formula (3-3) are as defined in formula (3).) The repeating structure CC is usually a repeating unit.

[0088] The amphoteric polymer 1 may contain a structure different from the repeating structures A to C. For example, the amphoteric polymer 1 may contain a repeating unit (hereinafter sometimes referred to as "another repeating unit of the amphoteric polymer 1") different from any of the repeating structure A (=repeating unit), the repeating unit containing the repeating structure B, and the repeating unit containing the repeating structure C.

[0089] Examples of monomers for forming other repeating units of amphoteric polymer 1 (hereinafter, may be referred to as "other monomers of amphoteric polymer 1") include the following: (meth)acrylic acid esters such as diethylaminoethyl (meth)acrylate, butyl (meth)acrylate, polyethylene glycol mono(meth)acrylate, glycerol (meth)acrylate, (meth)acryloyloxyethyl phosphate, (meth)acryloyloxyethyl phosphorylcholine, N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, methyl (meth)acrylate, glycidyl (meth)acrylate, dimethylaminoethyl (meth)acrylate, 2-hydroxyethyl methacrylate, and 2-methoxyethyl (meth)acrylate; and Radically polymerizable monomers such as acrylamide, N,N'-dimethylacrylamide, N-isopropyl(meth)acrylamide, (meth)acrylic acid, allyl alcohol, acrylonitrile, acrolein, vinyl acetate, sodium vinyl sulfonate, styrene, chlorostyrene, vinylphenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconic acid, itaconic acid esters, fumaric acid, fumaric acid esters, maleic acid, maleic acid esters, and maleic anhydride.

[0090] In this specification, "(meth)acrylic acid" basically means acrylic acid or methacrylic acid. When multiple "(meth)acrylic acids" can exist, "(meth)acrylic acid" means acrylic acid and / or methacrylic acid. Other terms similar to "(meth)acrylic acid" have the same meaning.

[0091] As the other monomers for amphoteric polymer 1, from the viewpoint of the solubility of amphoteric polymer 1, styrene, maleic anhydride, N,N'-dimethylacrylamide, butyl(meth)acrylate, and polyethylene glycol mono(meth)acrylate are preferred, and styrene and maleic anhydride are more preferred.

[0092] In order to fully utilize the performance of amphoteric polymer 1, the proportion of other repeating units in amphoteric polymer 1 is preferably 30 mol % or less, more preferably 10 mol % or less, and even more preferably 5 mol % or less, based on the total repeating units of amphoteric polymer 1. It is not necessary for amphoteric polymer 1 to contain other repeating units.

[0093] When amphoteric polymer 1 contains other repeating units (e.g., repeating units formed from styrene or repeating units formed from maleic anhydride), the proportion of the other repeating units of amphoteric polymer 1 is preferably 0.1 mol % or more, and more preferably 0.5 mol % or more, based on the total repeating units of amphoteric polymer 1, from the viewpoint of the solubility of amphoteric polymer 1 in water.

[0094] The amphoteric polymer 1 may be any of an alternating copolymer, a random copolymer, or a block copolymer, and may be, for example, a copolymer having an alternating copolymer structure or a random copolymer structure. From the viewpoint of ease of synthesis, the amphoteric polymer 1 is preferably an alternating copolymer or a random copolymer, and more preferably a random copolymer.

[0095] <Polyampholite (amphoteric polymer 2)> A preferred amphoteric polymer in one embodiment of the present invention is represented by formula (4):

[0096]

[0097] (In formula (4), * represents a bonding position, R ５ represents a hydrogen atom or a methyl group; ５ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), r represents an integer of 1 to 6, and X ２ is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３, and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position)) and a repeating structure D represented by formula (5):

[0098]

[0099] (In formula (5), * represents a bonding position, R ６ represents a hydrogen atom or a methyl group; ６ is absent or represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), s represents an integer of 0 to 6, and Y ２ is *-CO-O － , -S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S(O) ２ (O － ) (wherein * represents a bonding position, and C ６ H ４ represents a phenylene group.) represents a group selected from the group consisting of: and a polyampholite (sometimes referred to as "amphoteric polymer 2" in this specification) having a repeating structure E represented by the following formula:

[0100] A in formula (4) ５ *-CO-O-* and *-CO-NH-* are both A in formula (4). ５ A in formula (5) is bonded to the carbon atom (C) above at the "*-CO" side. ６ *-CO-O-* and *-CO-NH-* are both A in formula (4). ６ It is bonded to the carbon atom (C) above at the "*-CO" side.

[0101] Examples of the repeating structure D represented by the formula (4) include the following (in the formula, * represents a bonding position): ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ H ３ The repeating structure D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ３ The repeating structure D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ The repeating structure D, R ５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ H ３ The repeating structure D, R ５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ３ The repeating structure D, R ５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ The repeating structure D, R ５is a hydrogen atom, and A ５ is *-CO-NH-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a hydrogen atom, and A ５ is *-CO-NH-*, r is 2, and X ２ *-N ＋ H ３ a repeating structure D, and R ５ is a hydrogen atom, and A ５ is *-CO-NH-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ A repeating structure D is:

[0102] From the viewpoint of ease of synthesis of the amphoteric polymer 2, preferred examples of the repeating structure D include the following (in the following formula, * represents a bonding position): ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ H ３ The repeating structure D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ３ The repeating structure D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ The repeating structure D, R５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a hydrogen atom, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ H ３ The repeating structure D, R ５ is a hydrogen atom, and A ５ is *-CO-NH-*, r is 2, and X ２ *-N ＋ H ３ a repeating structure D, and R ５ is a hydrogen atom, and A ５ is *-CO-NH-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ A repeating structure D is:

[0103] From the viewpoint of ease of synthesis of the amphoteric polymer 2, more preferred examples of the repeating structure D include the following (in the following formula, * represents a bonding position): ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structures D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ H ３ The repeating structure D, R ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ３ a repeating structure D, and R５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-S ＋ (CH ３ ) ２ A repeating structure D is:

[0104] From the viewpoint of ease of synthesis of the amphoteric polymer 2, more preferred examples of the repeating structure D include the following (in the following formula, * represents a bonding position): ５ is a methyl group, and A ５ is *-CO-O-*, r is 2, and X ２ *-N ＋ (CH ３ ) ２ Repeating structure D which is H.

[0105] Examples of the repeating structure E represented by the formula (5) include the following (in the formula, * represents a bonding position, and C ６ H ４ represents a phenylene group.) : R ６ is a methyl group, and A ６ is absent, s is an integer from 0 to 3 (preferably 0), and Y ２ *-CO-O － The repeating structure E, R ６ is a hydrogen atom, and A ６ is absent, s is an integer from 0 to 3 (preferably 0), and Y ２ *-C ６ H ４ -S(O) ２ (O － ) repeating structures E, R ６ is a methyl group, and A ６ is *-CO-O-*, s is an integer of 0 to 3 (preferably 2), and Y ２ *-CO-O － The repeating structure E, R ６ is a methyl group, and A ６ is *-CO-O-*, s is an integer of 0 to 3 (preferably 2), and Y ２ *-S(O) ２ (O －) repeating structures E, R ６ is a methyl group, and A ６ is *-CO-O-*, s is an integer of 0 to 3 (preferably 2), and Y ２ is *-OP(O)(OH)(O － ), and R ６ is a hydrogen atom, and A ６ is absent, s is an integer from 0 to 3 (preferably 0), and Y ２ *-CO-O － A repeating structure E in which:

[0106] From the viewpoint of ease of synthesis of the amphoteric polymer 2, preferred examples of the repeating structure E include the following (in the following formula, * represents a bonding position, and C ６ H ４ represents a phenylene group.) : R ６ is a methyl group, and A ６ is absent, s is 0, and Y ２ *-CO-O － The repeating structure E, R ６ is a hydrogen atom, and A ６ is absent, s is 0, and Y ２ *-C ６ H ４ -S(O) ２ (O － ) repeating structures E, R ６ is a hydrogen atom, and A ６ is absent, s is 0, and Y ２ *-CO-O － A repeating structure E, and R ６ is a methyl group, and A ６ is *-CO-O-*, s is 2 or 3 (preferably 3), and Y ２ *-CO-O － A repeating structure E in which:

[0107] From the viewpoint of ease of synthesis of the amphoteric polymer 2, more preferred examples of the repeating structure E include the following (in the following formula, * represents a bonding position): ６ is a methyl group, and A ６is absent, s is 0, and Y ２ *-CO-O － The repeating structure E, R ６ is a hydrogen atom, and A ６ is absent, s is 0, and Y ２ *-CO-O － A repeating structure E, and R ６ is a methyl group, and A ６ is *-CO-O-*, s is 2 or 3 (preferably 3), and Y ２ *-CO-O － A repeating structure E in which:

[0108] From the viewpoint of ease of synthesis of the amphoteric polymer 2, more preferred examples of the repeating structure E include the following (in the following formula, * represents a bonding position): ６ is a methyl group, and A ６ is absent, s is 0, and Y ２ *-CO-O － A repeating structure E, and R ６ is a methyl group, and A ６ is *-CO-O-*, s is 3, and Y ２ *-CO-O － A repeating structure E in which:

[0109] From the viewpoint of ease of synthesis of the amphoteric polymer 2, in the formula (4), R ５ is a methyl group, and A ５ is *-CO-O-* (wherein * represents a bonding position), r is 2, and X ２ *-N ＋ (CH ３ ) ２ H (in the formula, * represents a bonding position), and ６ is a methyl group, and A ６ is absent or is *-CO-O-* (wherein * represents a bonding position), s is an integer of 0 to 3 (more preferably 0), and Y ２ *-CO-O － or *-S(O) ２ (O －) (wherein * represents the bonding position).

[0110] From the viewpoint of ease of synthesis of the amphoteric polymer 2, in the formula (4), R ５ is a methyl group, and A ５ is *-CO-O-* (wherein * represents a bonding position), r is 2, and X ２ *-N ＋ (CH ３ ) ２ H (in the formula, * represents a bonding position), and ６ is a methyl group, and A ６ is absent, s is 0, and Y ２ *-CO-O － (wherein * represents a bonding position) is more preferable.

[0111] From the viewpoints of achieving the cryoprotective effect of the amphoteric polymer 2 and cytotoxicity, the molar ratio of the repeating structure D to the sum of the repeating structures D and E (i.e., the amount (moles) of the repeating structure D / (the amount (moles) of the repeating structure D+the amount (moles) of the repeating structure E)) is preferably 0.1 or more and 0.9 or less. If the molar ratio is less than 0.1, the amphoteric polymer 2 becomes strongly anionic and is less likely to remain around cells, which may result in the cryoprotective effect not being exerted. If the molar ratio exceeds 0.9, the amphoteric polymer 2 becomes strongly cationic and may exhibit cytotoxicity. From the viewpoints of the cryoprotective effect and cytotoxicity of the amphoteric polymer 2, the molar ratio is more preferably 0.2 or more and 0.7 or less, even more preferably 0.2 or more and 0.6 or less, and particularly preferably 0.2 or more and 0.5 or less.

[0112] The amphoteric polymer 2 may contain a structure different from the repeating structures D and E. For example, the amphoteric polymer 2 may contain a repeating unit different from both the repeating structures D and E (hereinafter, this may be referred to as "another repeating unit of the amphoteric polymer 2"). Both the repeating structures D and E are usually repeating units.

[0113] Examples of monomers for forming other repeating units of amphoteric polymer 2 (hereinafter, may be referred to as "other monomers of amphoteric polymer 2") include the following: (meth)acrylic acid esters such as butyl (meth)acrylate, polyethylene glycol mono(meth)acrylate, glycerol (meth)acrylate, (meth)acryloyloxyethyl phosphate, (meth)acryloyloxyethyl phosphorylcholine, N-methylcarboxybetaine (meth)acrylate, N-methylsulfobetaine (meth)acrylate, methyl (meth)acrylate, glycidyl (meth)acrylate, 2-hydroxyethyl methacrylate, and 2-methoxyethyl (meth)acrylate; vinyl ethers such as methyl vinyl ether; and Various radical polymerizable monomers such as acrylamide, N,N'-dimethylacrylamide, N-isopropyl(meth)acrylamide, allyl alcohol, acrylonitrile, acrolein, vinyl acetate, sodium vinyl sulfonate, styrene, chlorostyrene, vinylphenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconic acid, itaconic acid esters, fumaric acid, fumaric acid esters, maleic acid, and maleic acid esters.

[0114] As other monomers for the amphoteric polymer 2, N,N'-dimethylacrylamide, butyl (meth)acrylate, and polyethylene glycol mono(meth)acrylate are preferred from the viewpoint of the solubility of the amphoteric polymer 2, with butyl (meth)acrylate being more preferred.

[0115] In order to fully utilize the performance of the amphoteric polymer 2, the proportion of the other repeating units in the amphoteric polymer 2 is preferably 30 mol % or less, and more preferably 10 mol % or less, based on the total repeating units of the amphoteric polymer 2. The amphoteric polymer 2 does not necessarily need to contain other repeating units.

[0116] When amphoteric polymer 2 contains other repeating units (e.g., repeating units formed from butyl (meth)acrylate), from the viewpoint of the cryoprotective effect of amphoteric polymer 2, the proportion of other repeating units of amphoteric polymer 2 is preferably 1 mol % or more, and more preferably 5 mol % or more, relative to the total repeating units of amphoteric polymer 2.

[0117] The amphoteric polymer 2 may be any of an alternating copolymer, a random copolymer, or a block copolymer, and may be, for example, a copolymer having an alternating copolymer structure or a random copolymer structure. From the viewpoint of ease of synthesis, the amphoteric polymer 2 is preferably a random copolymer.

[0118] <Zwitterionic Polymer> Examples of the combination of cationic groups and anionic groups that the zwitterionic polymer has include the following (in the following formula, * represents a bonding position): *-N ＋ (CH ３ ) ３ and *-O-P(O)(O － )-O-* combination, *-N ＋ (CH ３ ) ２ -* and *-CO-O － *-N ＋ (CH ３ ) ２ -* and *-S(O) ２ (O － ) combination, *-N ＋ (CH ３ ) ２ -* and *-O-P(O)(OH)(O － ) combination, *-S ＋ (CH ３ ) ２ and *-CO-O － combinations, and *-S ＋ (CH ３ ) ２ and *-S(O) ２ (O － ) combination.

[0119] From the viewpoint of ease of synthesis of the zwitterionic polymer, preferred combinations of cationic groups and anionic groups in the zwitterionic polymer include, for example, the following (in the following formula, * represents a bonding position): *-N ＋ (CH ３ ) ３ and *-O-P(O)(O － )-O-* combination, *-N ＋ (CH ３ ) ２ -* and *-CO-O － *-N ＋ (CH ３ ) ２ -* and *-S(O) ２ (O － ) combination, and *-S ＋ (CH ３ ) ２ and *-CO-O － A combination of.

[0120] From the viewpoint of ease of synthesis of the zwitterionic polymer, more preferred combinations of cationic groups and anionic groups in the zwitterionic polymer include, for example, the following (in the following formula, * represents a bonding position): *-N ＋ (CH ３ ) ３ and *-O-P(O)(O － )-O-* combination, and *-S ＋ (CH ３ ) ２ and *-CO-O － A combination of.

[0121] <Zwitterionic Polymer (Amphoteric Polymer 3)> A preferred amphoteric polymer in one embodiment of the present invention is a polymer represented by formula (6):

[0122] (In formula (6), * represents a bonding position), or a group represented by formula (7):

[0123]

[0124] (In formula (7), * represents a bonding position.) The zwitterionic polymer (sometimes referred to as "amphoteric polymer 3" in this specification) has a group represented by the following formula:

[0125] The amphoteric polymer 3 may contain a repeating unit (hereinafter, sometimes referred to as "another repeating unit of the amphoteric polymer 3") that is different from both the repeating unit having the group represented by the formula (6) and the repeating unit having the group represented by the formula (7).

[0126] Examples of monomers for forming other repeating units of amphoteric polymer 3 (hereinafter, may be referred to as "other monomers of amphoteric polymer 3") include the following: (meth)acrylic acid esters such as diethylaminoethyl (meth)acrylate, butyl (meth)acrylate, polyethylene glycol mono(meth)acrylate, glycerol (meth)acrylate, (meth)acryloyloxyethyl phosphate methyl (meth)acrylate, glycidyl (meth)acrylate, dimethylaminoethyl (meth)acrylate, 2-hydroxyethyl methacrylate, and 2-methoxyethyl (meth)acrylate; vinyl ethers such as methyl vinyl ether; and Various radical polymerizable monomers such as acrylamide, N,N'-dimethylacrylamide, N-isopropyl(meth)acrylamide, (meth)acrylic acid, allyl alcohol, acrylonitrile, acrolein, vinyl acetate, sodium vinyl sulfonate, styrene, chlorostyrene, vinylphenol, vinyl cinnamate, vinyl chloride, vinyl bromide, butadiene, vinylene carbonate, itaconic acid, itaconic acid esters, fumaric acid, fumaric acid esters, maleic acid, and maleic acid esters.

[0127] As other monomers for the amphoteric polymer 3, from the viewpoint of the solubility of the amphoteric polymer 3, N,N'-dimethylacrylamide, butyl (meth)acrylate, and polyethylene glycol mono(meth)acrylate are preferred, with butyl (meth)acrylate being more preferred.

[0128] In order to fully utilize the performance of the amphoteric polymer 3, the proportion of the other repeating units in the amphoteric polymer 3 is preferably 50 mol % or less, and more preferably 40 mol % or less, based on the total repeating units of the amphoteric polymer 3. The amphoteric polymer 3 does not necessarily need to contain other repeating units.

[0129] When other repeating units of the amphoteric polymer 3 (e.g., repeating units formed from butyl (meth)acrylate) are contained in the amphoteric polymer 3, from the viewpoint of the cryoprotective effect of the amphoteric polymer 3, the proportion of the other repeating units of the amphoteric polymer 3 is preferably 1 mol % or more, and more preferably 5 mol % or more, relative to the total repeating units of the amphoteric polymer 3.

[0130] The amphoteric polymer 3 may be any of an alternating copolymer, a random copolymer, or a block copolymer, and may be, for example, a copolymer having an alternating copolymer structure or a random copolymer structure. From the viewpoint of ease of synthesis, the amphoteric polymer 3 is preferably a random copolymer.

[0131] <Number-average molecular weight of amphoteric polymer> From the viewpoint of the solubility and cryoprotective effect of the amphoteric polymer, the number-average molecular weight of the amphoteric polymer is preferably 1,000 to 2,000,000, and more preferably 5,000 to 1,000,000. If this number-average molecular weight is less than 1,000, the cryoprotective effect of the amphoteric polymer may be weakened, and if this number-average molecular weight is more than 2,000,000, the solubility of the amphoteric polymer in water may be reduced. This number-average molecular weight is a value calculated by gel permeation chromatography using polyethylene glycol as a standard.

[0132] <Content of Amphoteric Polymer> The content of the amphoteric polymer is not particularly limited as long as it has a cryoprotective effect, but in order to achieve a good cryoprotective effect, the content is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, based on the total cryoprotective agent of the present invention. Furthermore, from the viewpoint of the solubility of the amphoteric polymer in water, the content is preferably 20% by mass or less, more preferably 10% by mass or less, based on the total cryoprotective agent of the present invention.

[0133] [Hydrophobic Amino Acids] Examples of hydrophobic amino acids include glycine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and ectoine. In one embodiment of the present invention, from the viewpoint of cryoprotective effect, the hydrophobic amino acid is preferably at least one selected from the group consisting of proline, ectoine, and valine, more preferably proline or ectoine. In another embodiment of the present invention, from the viewpoint of cryoprotective effect, the hydrophobic amino acid is preferably at least one selected from the group consisting of proline and valine, more preferably proline. The hydrophobic amino acid may be either D- or L-isomer.

[0134] The content of the hydrophobic amino acid is not particularly limited as long as it has a cryoprotective effect, but in order to achieve a good cryoprotective effect, it is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, based on the total cryoprotective agent of the present invention. Furthermore, from the viewpoint of the solubility of the hydrophobic amino acid in water, the content is preferably 10% by mass or less, more preferably 5% by mass or less, based on the total cryoprotective agent of the present invention.

[0135] [Water-soluble Cellulose] Examples of water-soluble cellulose include methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, and carboxymethyl cellulose.

[0136] From the viewpoint of cryoprotective effect, the water-soluble cellulose is preferably at least one selected from the group consisting of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, and carboxymethyl cellulose, more preferably at least one selected from the group consisting of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose, and even more preferably methyl cellulose.

[0137] From the viewpoint of ease of handling of the water-soluble cellulose, the viscosity of an aqueous solution having a concentration of 2% by mass of water-soluble cellulose at 20°C is preferably 0.1 mPa·s or more, more preferably 1 mPa·s or more, even more preferably 10 mPa·s or more, and preferably 10,000 mPa·s or less, more preferably 1,000 mPa·s or less, even more preferably 100 mPa·s or less. The viscosity is a value measured using a rotational viscometer.

[0138] The content of water-soluble cellulose is not particularly limited as long as it has a cryoprotective effect, but in order to achieve a good cryoprotective effect, it is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, based on the total cryoprotectant of the present invention. Furthermore, from the viewpoint of the solubility of water-soluble cellulose in water, the content is preferably 5% by mass or less, more preferably 1% by mass or less, based on the total cryoprotectant of the present invention.

[0139] [Water] The cryoprotectant of the present invention may contain water. Preferred examples of water include purified water, pure water, and ion-exchanged water. When the cryoprotectant of the present invention contains water, from the viewpoint of the cryoprotective effect, the water content is preferably 5% by mass or more, more preferably 10% by mass or more, even more preferably 50% by mass or more, and particularly preferably 60% by mass or more, and is preferably 90% by mass or less, and more preferably 80% by mass or less, based on the total amount of the cryoprotectant of the present invention.

[0140] [Culture Medium] The cryoprotectant of the present invention may contain a culture medium. The culture medium is not particularly limited as long as it is suitable for the cells to be cryopreserved, and a general cell culture medium can be used. Examples of the culture medium include Dulbecco's Modified Eagle's Medium (DMEM), α-MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, and GMEM medium. A mixture of these culture media can also be used. Furthermore, the culture medium may be supplemented with human serum (HSA), bovine serum (e.g., fetal bovine serum (FBS)), glutamine, or antibiotics, as needed.

[0141] [Other Components] The present invention may contain other components. Examples of other components include reagents, organic solvents, sugars, polysaccharides, proteins, salts, surfactants, buffer solutions, polyvinyl alcohol, and the like that are commonly used in this field for the purpose of improving the survival rate of cells after freezing and thawing. Each of the other components may be used alone, or two or more may be used in combination.

[0142] Examples of organic solvents include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, etc. From the viewpoint of cryoprotective effect, DMSO is preferred.

[0143] When the cryoprotectant of the present invention contains DMSO, the content of DMSO is preferably 1% by mass or more, more preferably 5% by mass or more, relative to the entire cryoprotectant of the present invention, from the viewpoint of cryoprotective effect, and is preferably 30% by mass or less, more preferably 20% by mass or less, from the viewpoint of cytotoxicity.

[0144] Examples of sugars include lactose, sucrose, trehalose, etc. Examples of polysaccharides include alginic acid, pullulan, chitosan, dextran, glucomannan, hydroxyethyl starch, etc.

[0145] Examples of proteins include proteins in bovine serum (e.g., bovine serum albumin), proteins in fetal bovine serum (FBS), antifreeze peptides, gelatin, casein, proteins in human serum (e.g., human serum albumin), growth factors, StemFit (registered trademark, manufactured by Ajinomoto Healthy Supply Co., Inc.), and the like.

[0146] When the cryoprotectant of the present invention contains FBS, the content of FBS relative to the entire cryoprotectant of the present invention is preferably 5% by mass or more, more preferably 10% by mass or more, and even more preferably 30% by mass or more, from the viewpoint of cryoprotective effect, and is preferably 80% by mass or less, more preferably 60% by mass or less, and even more preferably 50% by mass or less, from the viewpoint of ease of handling.

[0147] Examples of salts include salts of inorganic acids such as phosphates, borates, sulfates and tris hydrochlorides, and salts of organic acids such as acetic acid, citric acid, malic acid, maleic acid and gluconic acid.

[0148] Examples of surfactants include polyoxyethylene alkyl ether, Tween 20, and Pluronic (registered trademark).

[0149] As the buffer, any buffer normally used in this field can be used as long as it does not destroy the physiological activities of the protein, such as enzymatic activity and antigenicity. Examples of buffers include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, etc. Two or more buffers may be mixed and used.

[0150] [Method for Cryopreserving Cells] The present invention provides a method for cryopreserving cells (sometimes referred to herein as the "method of the present invention").

[0151] The method of the present invention comprises: step 1 of mixing cells with the cryoprotectant of the present invention; and step 2 of freezing the mixture containing the cells and the cryoprotectant.

[0152] <Step 1> Step 1 is a step of mixing cells with the cryoprotectant of the present invention. For the mixing, a means commonly used in the field of cell handling can be used. ４ From the viewpoint of cryoprotective effect, the amount of the cryoprotectant of the present invention used per cell is preferably 1 to 2,000 μL, more preferably 2 to 1,500 μL, and even more preferably 5 to 1,000 μL.

[0153] <Step 2> Step 2 is a step of freezing a mixture containing cells and a cryoprotectant. From the viewpoint of suppressing cell damage, the cooling rate for freezing is preferably 0.5 to 10°C / min, more preferably 0.5 to 2°C / min. Furthermore, the cryopreservation temperature after cooling is, for example, −80°C or lower. From the viewpoint of more stable cryopreservation, it is preferable to store the frozen mixture in liquid nitrogen or liquid nitrogen vapor.

[0154] [Method for cryopreserving two-dimensionally cultured cells] A preferred embodiment of the method of the present invention is a method for cryopreserving two-dimensionally cultured cells (sometimes referred to as "Method 1 of the present invention" in this specification).

[0155] Method 1 of the present invention comprises: Step A: two-dimensionally culturing adherent cells in a medium on a culture substrate; Step C: mixing the two-dimensionally cultured cells with the cryoprotectant of the present invention; and Step D: freezing the mixture containing the two-dimensionally cultured cells on the culture substrate and the cryoprotectant of the present invention. That is, Method 1 of the present invention is an embodiment of the method of the present invention in which the "cells" of the method of the present invention are the "two-dimensionally cultured cells," the method comprises Step A before Step 1, Step 1 is Step C, and Step 2 is Step D.

[0156] <Step A> Step A is a step of two-dimensionally culturing adherent cells in a medium on a culture substrate. As a method for two-dimensional culture, a method commonly used in the field of cell culture can be used. The medium is not particularly limited, and for example, the medium described above can be used.

[0157] The material of the culture substrate is not particularly limited, and examples thereof include plastic, metal, silicone, glass, etc. Only one type of culture substrate material may be used, or two or more types may be used in combination. Examples of materials suitable for cell culture include silicone, glass, and plastic that are resistant to water and organic solvents. Examples of plastics include acrylic polymers such as polymethyl methacrylate, polystyrene, polyethylene terephthalate, polycarbonate, etc. Examples of silicones include silicone rubber such as polydimethylsiloxane, etc.

[0158] The shape of the culture substrate may be, for example, a vessel-like, plate-like, or fibrous shape, or may be a porous shape with holes or grooves. Among these, vessel-like, plate-like, and fibrous shapes are preferred from the viewpoint of ease of culturing two-dimensionally cultured cells, and vessel-like shapes such as petri dishes and flasks are more preferred.

[0159] <Step B (Step of Removing Culture Supernatant)> Step B, in which the culture supernatant is removed from the two-dimensionally cultured cells, may be carried out between Step A and Step C. As used herein, "culture supernatant" refers to the "supernatant obtained when cells are cultured in a medium." The means for removing the culture supernatant is not particularly limited as long as it does not adversely affect the growth of the two-dimensionally cultured cells. For example, the culture supernatant can be removed by suction with a pipette or aspirator, by inversion, or by centrifugation.

[0160] <Step C> Step C is a step of mixing the two-dimensionally cultured cells with the cryoprotectant of the present invention. For the mixing, a means commonly used in the field of cell culture can be used. ４ From the viewpoint of cryoprotective effect, the amount of the cryoprotectant of the present invention used per two-dimensionally cultured cell is preferably 10 to 300 μL, more preferably 20 to 200 μL, even more preferably 50 to 200 μL, and particularly preferably 50 to 100 μL.

[0161] <Step D> Step D is a step of freezing a mixture containing two-dimensionally cultured cells on a culture substrate and the cryoprotectant of the present invention. From the viewpoint of suppressing cell damage, the cooling rate for freezing is preferably 0.5 to 10°C / min, more preferably 0.5 to 2°C / min. Furthermore, the cryopreservation temperature after cooling is, for example, −80°C or lower. From the viewpoint of more stable cryopreservation, it is preferable to store the frozen mixture in liquid nitrogen or liquid nitrogen vapor.

[0162] [Method for cryopreserving three-dimensionally cultured cells] A preferred embodiment of the method of the present invention is a method for cryopreserving three-dimensionally cultured cells (sometimes referred to as "Method 2 of the present invention" in this specification).

[0163] Method 2 of the present invention comprises: Step E of three-dimensionally culturing cells in a medium; Step G of mixing the three-dimensionally cultured cells with the cryoprotectant of the present invention; and Step H of freezing the mixture containing the three-dimensionally cultured cells and the cryoprotectant of the present invention. That is, Method 2 of the present invention is an embodiment of the method of the present invention in which the "cells" of the method of the present invention are the "three-dimensionally cultured cells," the method comprises Step E before Step 1, Step 1 is Step G, and Step 2 is Step H.

[0164] <Step E> Step E is a step of three-dimensionally culturing cells in a culture medium. As a three-dimensional culture method, a method commonly used in the field of cell culture can be used. There is no particular limitation on the culture medium, and for example, the above-mentioned medium can be used. As a method for producing spheroids, for example, a method using a non-adhesive culture substrate, a hanging drop method, etc. can be mentioned. Furthermore, as a method for producing organoids, for example, a method of embedding cells in a scaffold material such as Matrigel, etc. can be mentioned.

[0165] <Step F (Step of Removing Culture Supernatant)> Step F, in which the three-dimensionally cultured cells are transferred to a cryopreservation container and the culture supernatant is removed, may be performed between steps E and G. The means for removing the culture supernatant is not particularly limited as long as it does not damage the three-dimensionally cultured cells. For example, the culture supernatant can be removed by suction removal with a pipette or aspirator, by inversion, or by centrifugation.

[0166] <Step G> Step G is a step of mixing the 3D cultured cells with the cryoprotectant of the present invention. For the mixing, a method commonly used in this field can be used. ４ From the viewpoint of cryoprotective effect, the amount of the cryoprotectant of the present invention used per 3D-cultured cell is preferably 100 to 2,000 μL, more preferably 200 to 1,500 μL, and even more preferably 500 to 1,000 μL.

[0167] <Step H> Step H is a step of freezing a mixture containing three-dimensionally cultured cells and the cryoprotectant of the present invention. From the viewpoint of suppressing cell damage, the cooling rate for freezing is preferably 0.5 to 10°C / min, more preferably 0.5 to 2°C / min. Furthermore, the cryopreservation temperature after cooling is, for example, −80°C or lower. From the viewpoint of more stable cryopreservation, it is preferable to store the frozen mixture in liquid nitrogen or liquid nitrogen vapor.

[0168] [Method for cryopreserving cells suspended in a medium] A preferred embodiment of the method of the present invention is a method for cryopreserving cells suspended in a medium (sometimes referred to as "Method 3 of the present invention" in this specification).

[0169] Method 3 of the present invention (i.e., a method for cryopreserving cells suspended in a medium) comprises: Step I of suspending cells in a medium; Step K of mixing the cells with a cryoprotectant; and Step L of freezing the mixture containing the cells and the cryoprotectant. That is, Method 3 of the present invention is an embodiment of the method of the present invention in which the "method for cryopreserving cells" of the present invention is the "method for cryopreserving cells suspended in a medium" described above, and comprises Step I before Step 1, Step 1 being Step K, and Step 2 being Step L.

[0170] <Step I> Step I is a step of suspending cells in a medium. There is no particular limitation on the method used for this, and methods commonly used in the field of cell handling can be used. There is no particular limitation on the medium, and for example, the medium described above can be used. The cells may be adherent cells. When adherent cells are used, methods commonly used in the field of cell culture can be used to detach the adherent cells from the culture substrate. Examples of such methods include, but are not limited to, a method using a protease such as trypsin, and a physical detachment method using a cell scraper or the like. The amount of cells per mL of medium is preferably 1 x 10 ４ ~1 x 10 ８ cells, more preferably 1 x 10 ５ ~1 x 10 ７ cells.

[0171] <Step J (Step of Removing Supernatant)> Step J of removing the supernatant from the mixture containing the medium and the cells may be performed between Step I and Step K. The means for removing the supernatant is not particularly limited as long as it does not damage the cells suspended in the medium. For example, the supernatant can be removed by suction with a pipette or aspirator, by inversion, or by centrifugation.

[0172] <Step K> Step K is a step of mixing cells with the cryoprotectant of the present invention. For the mixing, a method commonly used in the field of cell handling can be used. ４ From the viewpoint of cryoprotective effect, the amount of the cryoprotectant of the present invention used per suspended cell is preferably 1 to 20 μL, more preferably 2 to 15 μL, and even more preferably 5 to 10 μL.

[0173] <Step L> Step L is a step of freezing a mixture containing cells and the cryoprotectant of the present invention. From the viewpoint of suppressing cell damage, the cooling rate for freezing is preferably 0.5 to 10°C / min, more preferably 0.5 to 2°C / min. Furthermore, the cryopreservation temperature after cooling is, for example, −80°C or lower. From the viewpoint of more stable cryopreservation, it is preferable to store the frozen mixture in liquid nitrogen or liquid nitrogen vapor.

[0174] [Thawing] The method for thawing a frozen mixture containing cells (e.g., two-dimensionally cultured cells, three-dimensionally cultured cells, or cells suspended in a medium) and the cryoprotectant of the present invention is not particularly limited, and any method commonly used in this field can be used. In order to shorten the thawing time, it is preferable to temporarily thaw the mixture at a temperature in the range of -80°C to 0°C, add a culture medium, and then warm the mixture.

[0175] The culture medium used in the thawing method preferably contains bovine serum (e.g., fetal bovine serum (FBS)) and / or human serum. The culture medium used in the thawing method or for cell culture after thawing may contain an antioxidant and / or an apoptosis inhibitor to accelerate cell recovery from freezing stress. Examples of antioxidants include, but are not limited to, N-acetyl-L-cysteine, vitamin A, vitamin C, vitamin E, flavonoids, and derivatives thereof. Examples of apoptosis inhibitors include, but are not limited to, ROCK inhibitors (e.g., Y27632, AZD-5363, Fasudil, GSK-429286A, H89, Pantoprazole, RKI-1447, Thiazovivin, etc.).

[0176] The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples.

[0177] <Synthesis Example 1: Synthesis of Polymer A>

[0178]

[0179] 2 g of poly(methyl vinyl ether-alt-maleic anhydride) (hereinafter abbreviated as "pMal", manufactured by Sigma-Aldrich, number average molecular weight: 80,000) was weighed into a four-necked flask, and 40 mL of tetrahydrofuran (hereinafter abbreviated as "THF") was added. The mixture was stirred at 50°C to dissolve the mixture. Four equivalents of N,N-dimethylethylenediamine relative to the maleic anhydride moiety were added thereto, and the mixture was stirred at 50°C to react. After 1 hour of reaction, the solid was collected by suction filtration. The mixture was then dialyzed against a saturated aqueous solution of sodium bicarbonate and ion-exchanged water (molecular weight cutoff (hereinafter abbreviated as "MWCO"): 1000), and freeze-dried to obtain Polymer A having the above structure. N,N-dimethylethylenediamine introduction rate: 100% ( １ (Assigned by H NMR) Polymer A １ H NMR (DCl+D ２ O, 400MHz) δ 3.4-3.5ppm (-O-CH ３ ), 2.7-3.1ppm (-CONH-CH ２ -CH ２-), 2.4-2.6ppm (-N(CH ３ ) ２ ), 0.9-1.9ppm (-CH ２ -CH(O-CH ３ )-), 1.9-4.0 ppm (polymer main chain) Yield of polymer A: 2.39 g

[0180] The number-average molecular weight and polydispersity (weight-average molecular weight / number-average molecular weight) of the resulting polymer A were determined by gel permeation chromatography using a differential refractometer as a detector. A JASCO PU-2080 pump, a JASCO RI-2031 detector (differential refractometer), and a UV-2075 detector (UV) were used. Two columns, a TSKGel G3000PWXL and a TSKGel G5000PWXL (column size: 4.6 mm x 25 cm) manufactured by Tosoh Corporation, were connected together. The developing solvent was 0.1 M phosphate buffer adjusted to pH 7.4. The measurement conditions were a flow rate of 0.6 mL / min, a column temperature of 40°C, a sample concentration of 2 mg / mL, and an injection volume of 20 μL. Polyethylene glycol was used as a standard. As a result of gel permeation chromatography, the number average molecular weight of polymer A was 82,600 and its polydispersity index was 2.78.

[0181] Synthesis Example 2: Synthesis of polymer B (1) Synthesis of polymer b Polymer b used in the synthesis of polymer B was synthesized as follows.

[0182]

[0183] Monomer m1 represented by the above formula was synthesized in the same manner as in Synthesis Example 1 described in JP-A 2002-173351. The obtained monomer m1 (1524 g, 1 mol), maleic anhydride (147 g, 1.5 mol), styrene (52 g, 0.5 mol), and toluene (300 g) were weighed out, and the monomers were copolymerized using benzoyl peroxide (4.84 g) dissolved in toluene (96.8 g) as an initiator. After copolymerization, the toluene was distilled off to obtain polymer b having the repeating unit. The molar ratio of the repeating units: x / y / z=49.6 / 49.6 / 0.8 ( １ (Assigned by H NMR) Polymer b１ H NMR (CD ３ OD, 400MHz) δ 3.6-3.7ppm (-O-CH ２ -CH ２ -O-), 3.4ppm (-O-CH ３ ), 1.0-2.3ppm (-CH ２ -CH-), 2.3-3.9 ppm (polymer main chain)

[0184] The number average molecular weight of the obtained polymer b was measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer b was 10,000.

[0185] (2) Synthesis of Polymer B Using the obtained polymer b, polymer B was synthesized as follows.

[0186]

[0187] 2 g of polymer b (number average molecular weight: 10,000) was weighed into a four-neck flask, and 20 mL of THF was added. The mixture was stirred at 50°C to dissolve the polymer. Four equivalents of N,N-dimethylethylenediamine and triethylamine relative to the maleic anhydride units were added thereto, and the mixture was stirred at 50°C to react. After 4 hours of reaction, 100 mg of Kyoward 600 was added to the reaction mixture, and the resulting mixture was stirred at room temperature (23°C) for 1 hour and then suction filtered. After dialysis against saturated aqueous sodium bicarbonate and ion-exchanged water (MWCO: 1000), the mixture was freeze-dried to obtain polymer B having the repeating units described above. N,N-dimethylethylenediamine introduction rate: 61.6% ( １ (Assigned by H NMR) Polymer B １ H NMR (DC1+CD ３ OD, 400MHz) δ 3.4-3.9ppm (-COO-CH ２ -CH ２ --, --O-CH ２ -CH ２ -O-), 3.3-3.4ppm (-O-CH ３ ), 2.9-3.2ppm (-N-(CH ３ ) ２ ), 1.0-2.4ppm (-CH ２-CH-), 2.3-3.9 ppm (polymer main chain) Molar ratio of repeating units: x / y1 / y2 / z=49.6 / 19.0 / 30.6 / 0.8 ( １ H NMR) Yield of Polymer B: 1.67 g

[0188] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer B were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer B was 17,300, and the polydispersity was 3.34.

[0189] Synthesis Example 3: Synthesis of Polymer C

[0190]

[0191] 2 g of pMal (Sigma-Aldrich, number average molecular weight: 80,000) was weighed into a four-neck flask, 40 mL of THF was added, and the mixture was stirred at 50°C to dissolve the product. 2-Methylthioethanol and triethylamine were added in amounts of 4 equivalents and 4.8 equivalents, respectively, relative to the maleic anhydride moiety, and the mixture was stirred at 50°C to allow the reaction to proceed. After the reaction for 1 hour, the mixture was subjected to suction filtration to recover the solid. The solid was then dialyzed (MWCO: 1000) against a saturated aqueous solution of sodium bicarbonate and ion-exchanged water, and then freeze-dried.

[0192] 500 mg of the obtained dried product was weighed into a four-necked flask, and 4 mL of ion-exchanged water and 7 mL of ethanol were added and dissolved at room temperature. After 10 minutes of nitrogen bubbling (60 mL / min), 500 μL (4.5 equivalents) of iodomethane was added, and the reaction was carried out at room temperature for 19 hours. After that, the product was dialyzed against ion-exchanged water (MWCO: 1000) and freeze-dried to obtain polymer C having the above structure. 2-Methylthioethanol incorporation rate: 100% ( １ (Assigned by H NMR) Polymer C １ H NMR (D ２ O, 400MHz) δ 4.3-4.6ppm (-COO-CH ２ -), 3.5-3.7ppm (-CH ２ -CH ２ -), 3.1-3.3 ppm (-O-CH ３ ), 2.8-2.9ppm (-S-(CH ３ )２ ), 1.3-2.1ppm (-CH ２ -CH-), 2.3-3.9 ppm (polymer main chain) Yield of polymer C: 0.419 g

[0193] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer C were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer C was 83,000, and the polydispersity was 2.80.

[0194] <Synthesis Example 4: Synthesis of Polymer D>

[0195]

[0196] 665 mg of dimethylaminoethyl methacrylate and 364 mg of methacrylic acid were weighed and dissolved in 4.2 mL of ion-exchanged water. Also, 63 mg of butyl methacrylate, 7.5 mg of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, and 1 mg of 4,4'-azobis(4-cyanovaleric acid) were weighed and dissolved in 2.8 mL of ethanol. The two solutions were mixed in a four-neck flask, and after 15 minutes of nitrogen bubbling (20 mL / min), the nitrogen flow was switched to (15 mL / min), and the reaction was carried out at 70°C for 20 hours. The resulting mixture was then dialyzed against a 50 / 50 volumetric ratio of ion-exchanged water / ethanol mixture for one day and against ion-exchanged water for two days (MWCO: 1000), followed by lyophilization to obtain Polymer D having the repeating units described above. The molar ratio of the repeating units was x / y / z=48 / 45 / 7 ( １ (Assigned by H NMR) Polymer D １ H NMR (D ２ O, 400 MHz) δ 4.2-4.5 ppm (2-dimethylaminoethyl methacrylate unit (hereinafter abbreviated as "DMAEMA"): -COO-CH ２ -), 4.0-4.2 ppm (butyl methacrylate unit (hereinafter abbreviated as "BMA"): -COO-CH ２ -), 3.3-3.7ppm (-CH ２ -CH ２ -), 2.7-3.2ppm (-N-(CH ３ ) ２ ), 0.5-2.5ppm (BMA:-CH２ -CH ２ -CH ３ , polymer backbone) Yield of polymer D: 0.700 g

[0197] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer D were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer D was 31,600, and the polydispersity was 1.20.

[0198] Synthesis Example 5: Synthesis of Polymer E

[0199]

[0200] 411 mg of dimethylaminoethyl methacrylate and 643 mg of 3-sulfopropyl methacrylate potassium salt were weighed out and dissolved in 4.2 mL of ion-exchanged water. Also, 39 mg of butyl methacrylate, 7.5 mg of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, and 1 mg of 4,4'-azobis(4-cyanovaleric acid) were weighed out and dissolved in 1.8 mL of ethanol. The two solutions were mixed in a four-necked flask, and after 15 minutes of nitrogen bubbling (20 mL / min), the nitrogen flow was switched to (15 mL / min) and the reaction was carried out at 70°C for 20 hours. The resulting mixture was then dialyzed against a 50 / 50 volumetric ratio of ion-exchanged water / ethanol mixture for one day and against ion-exchanged water for two days (MWCO: 1000), followed by lyophilization to obtain Polymer E having the repeating units described above. The molar ratio of the repeating units: x / y / z = 23 / 69 / 8 ( １ (Assigned by H NMR) Polymer E １ H NMR (D ２ O, 400MHz) δ 4.2-4.5ppm (DMAEMA:-COO-CH ２ -), 4.0-4.2 ppm (3-sulfopropyl methacrylate unit (hereinafter abbreviated as "SPMA"): -COO-CH ２ -, BMA:-COO-CH ２ -), 3.3-3.7ppm (DMAEMA:-CH ２ -CH ２ -), 2.7-3.2ppm (DMAEMA:-N-(CH ３ ) ２ , SPMA:-CH２ -CH ２ -CH ２ -), 2.0-2.3ppm (SPMA:-CH ２ -CH ２ -CH ２ -), 0.5-2.5ppm (BMA:-CH ２ -CH ２ -CH ３ , polymer backbone) Yield of Polymer E: 0.66 g

[0201] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer E were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer E was 11,100, and the polydispersity was 1.37.

[0202] Synthesis Example 6: Synthesis of Polymer F

[0203]

[0204] 975 mg of (2-methacryloyloxy)ethyl phosphorylcholine was weighed out and dissolved in 3.2 mL of ion-exchanged water. 117 mg of butyl methacrylate, 7.5 mg of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, and 1 mg of 4,4'-azobis(4-cyanovaleric acid) were weighed out and dissolved in 0.8 mL of ethanol. The two solutions were mixed in a four-necked flask, and after 15 minutes of nitrogen bubbling (20 mL / min), the nitrogen flow was switched to (20 mL / min) and the reaction was carried out at 70°C for 3 hours. The mixture was then dialyzed against a 50 / 50 volumetric ratio of ion-exchanged water / ethanol mixture for 1 day and then against ion-exchanged water for 2 days (MWCO: 1000), followed by lyophilization to obtain Polymer F having the repeating unit described above. The molar ratio of repeating units: x / y = 88 / 12 ( １ (Assigned by H NMR) Polymer F １ H NMR (D ２ O, 400 MHz) δ 3.9-4.4 ppm ((2-methacryloyloxy)ethylphosphorylcholine unit: -COO-CH ２ -CH ２ -, -PO-CH ２ -, BMA:-COO-CH ２ -), 3.6-3.8ppm (-CH２ -CH ２ -N-), 1.6-1.8ppm (BMA:-CH ２ -CH ２ -CH ３ ), 1.4-1.6ppm (BMA:-CH ２ -CH ２ -CH ３ ), 1.0-1.2ppm (BMA:-CH ２ -CH ２ -CH ３ ), 0.5-2.5 ppm (polymer main chain) Yield of polymer F: 0.75 g

[0205] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer F were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer F was 23,700, and the polydispersity was 1.25.

[0206] Synthesis Example 7: Synthesis of polymer G (1) Synthesis of N-(prop-2-enoyl)-L-methionine N-(prop-2-enoyl)-L-methionine used in the synthesis of polymer G was synthesized as follows.

[0207]

[0208] L-methionine methyl ester (10.0 g, 50.0 mmol) was placed in a four-neck flask and dissolved in 100 mL of THF. The reaction solution was cooled to 0°C, and triethylamine (15 mL, 108 mmol) and acrylic acid chloride (5.4 mL, 60 mmol) were added dropwise over 15 minutes. The reaction solution was then warmed to room temperature and stirred for 1 hour. 2 mL of methanol was added to the reaction solution, and the mixture was stirred at room temperature for an additional 1 hour. After the solvent was evaporated under reduced pressure, 200 mL of methyl tert-butyl ether (hereinafter abbreviated as "MTBE") was added. The organic phase was washed twice with 50 mL of saturated aqueous ammonium chloride solution, once with 50 mL of saturated aqueous sodium bicarbonate solution, and once with 100 mL of saturated saline, then dried over magnesium sulfate, and the solvent was evaporated under reduced pressure.

[0209] The obtained concentrate was dissolved in 50 mL of acetonitrile, and 40 mL of 1N aqueous sodium hydroxide solution was added, followed by stirring at room temperature for 2 hours. The reaction solution was neutralized with 4N hydrochloric acid, and the solvent was evaporated under reduced pressure. The pH of the obtained concentrate was adjusted to 3, and then extracted with 150 mL of ethyl acetate. The organic phase was washed once with 100 mL of saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to obtain N-(prop-2-enoyl)-L-methionine. Yield of N-(prop-2-enoyl)-L-methionine: 5.02 g １ H NMR (CD ３ OD, 400MHz) δ 6.2-6.4ppm (CH ２ =CH,m,2H), 5.69ppm(CH ２ =CH, t, 1H), 4.63ppm (NH-CH-COOH, t, 1H), 2.5-2.6ppm (CH ２ -CH ２ -S, m, 2H), 2.0-2.2ppm (CH ２ -CH ２ -S, m, 2H), 2.01 ppm (S-CH ３ , s, 3H)

[0210] (2) Synthesis of Polymer G Using the obtained N-(prop-2-enoyl)-L-methionine, Polymer G was synthesized as follows.

[0211]

[0212] 944 mg of N-(prop-2-enoyl)-L-methionine, 149 mg of butyl acrylate, 7.5 mg of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, and 1 mg of 4,4'-azobis(4-cyanovaleric acid) were weighed and dissolved in 4.8 mL of an N,N-dimethylformamide / ethyl acetate mixed solvent (volume ratio: 4 / 1). The resulting solution was stirred in a four-neck flask, and after 15 minutes of nitrogen bubbling (20 mL / min), the flow was switched to nitrogen (20 mL / min), and the reaction was carried out at 70°C for 20 hours. The polymer was then purified by reprecipitation using an MTBE / hexane mixed solvent (volume ratio: 50 / 50) as a poor solvent and then dried under reduced pressure to obtain the target precursor.

[0213] 400 mg of the precursor was dissolved in 4 mL of ethanol, and nitrogen bubbling (60 mL / min) was performed for 10 minutes. 400 μL of iodomethane was added to the resulting mixture, and the mixture was reacted at 23° C. for 20 hours. After the reaction, the mixture was dialyzed against a mixed solvent of ion-exchanged water / ethanol (volume ratio: 50 / 50) for 1 day and against ion-exchanged water for 2 days (MWCO: 1000), and then freeze-dried to obtain Polymer G having the repeating unit. The molar ratio of the repeating units: x / y=67 / 33 ( １ H NMR) Yield of polymer G: 0.19 g

[0214] The number average molecular weight and polydispersity (weight average molecular weight / number average molecular weight) of the obtained polymer G were measured in the same manner as in Synthesis Example 1. The number average molecular weight of polymer G was 5,910, and the polydispersity was 1.35.

[0215] <Synthesis Example 8: Synthesis of Polymer J>

[0216]

[0217] 20 g of pMal (Sigma-Aldrich, number average molecular weight: 80,000) was weighed into a four-neck flask and dissolved in 250 mL of THF. The resulting solution was added dropwise to 500 mL of MTBE and then filtered using Kiriyama Funnel Filter Paper No. 5C. After filtration, the solid was recovered by drying under reduced pressure. The above procedure was repeated twice to purify pMal.

[0218] 6.0 mL of N,N-dimethyl-1,3-propanediamine was added to a four-neck flask, and 63 mL of ion-exchanged water was added in an ice bath and mixed to prepare an aqueous solution. 5.0 g of purified pMal was weighed into a four-neck flask, and 50 mL of acetonitrile (hereinafter abbreviated as "ACN") was added and dissolved to prepare a pMal solution, which was then transferred to a dropping funnel. The pMal solution was added dropwise to the aqueous solution, and the mixture was reacted for 1 hour. After distilling off the ACN by vacuum concentration, 6 M hydrochloric acid was added to the residue to adjust the pH of the mixture to 6.0. After dialysis against ion-exchanged water (MWCO: 3,500) and freeze-drying, polymer J having the above structure was obtained. N,N-dimethyl-1,3-propanediamine introduction rate: 76% ( １ (Assigned by H NMR) Polymer J １ H NMR (NaOD+D ２ O, 400MHz) δ 2.9-3.9ppm (-O-CH ３ , -CONH-CH ２ -CH ２ -), 2.3-2.9ppm (-CH ２ -CH(O-CH ３ )-), 2.1-2.3ppm (-CONH-CH ２ -CH ２ -CH ２ -), 1.9-2.1ppm (-N(CH ３ ) ２ ), 1.3-1.7ppm (-CONH-CH ２ -CH ２ -CH ２ -), 1.3-4.0 ppm (polymer main chain) Molar ratio of repeating units: x / y = 76 / 24 ( １ H NMR) Yield of Polymer J: 5.3 g

[0219] The number-average molecular weight and polydispersity index (weight-average molecular weight / number-average molecular weight) of the obtained polymer J were determined by gel permeation chromatography using a differential refractometer as a detector. A JASCO PU-2080 pump, a JASCO RI-2031 detector (differential refractometer), and a UV-2075 detector (UV) were used. The column used consisted of two TSKGel G3000PWXL and TSKGel G5000PWXL columns (column size: 4.6 mm x 25 cm) connected together, both manufactured by Tosoh Corporation. The developing solvent was 0.1 M phosphate buffer adjusted to pH 7.4. The measurement conditions were a flow rate of 0.45 mL / min, a column temperature of 40°C, a sample concentration of 20 mg / mL, and an injection volume of 100 μL. Polyethylene glycol was used as the standard. As a result of gel permeation chromatography, the number average molecular weight of Polymer J was 46,400 and its polydispersity index was 2.78.

[0220] Synthesis Example 9: Synthesis of Polymer K

[0221]

[0222] 20 g of pMal (Sigma-Aldrich, number average molecular weight: 80,000) was weighed into a four-neck flask and dissolved in 250 mL of THF. The resulting solution was added dropwise to 500 mL of methyl methyl tertiary butyl ether (MTBE), followed by filtration using Kiriyama Funnel Filter Paper No. 5C. After filtration, the solid was recovered by drying under reduced pressure. The above procedure was repeated twice to purify pMal.

[0223] 6.2 mL of N,N-dimethyl-1,4-butanediamine was added to a four-neck flask, and 250 mL of ion-exchanged water was added in an ice bath and mixed to prepare an aqueous solution. 4.5 g of purified pMal was weighed into a four-neck flask, and 45 mL of ACN was added and dissolved to prepare a pMal solution, which was then transferred to a dropping funnel. The pMal solution was added dropwise to the aqueous solution, and the mixture was reacted for 1 hour. After distilling off the ACN by vacuum concentration, 6 M hydrochloric acid was added to the residue to adjust the pH of the mixture to 6.0. After dialysis against ion-exchanged water (MWCO: 3,500) and freeze-drying, polymer K having the above structure was obtained. N,N-dimethyl-1,4-butanediamine introduction rate: 75% ( １(Assigned by H NMR) Polymer K １ H NMR (NaOD+D ２ O, 400MHz) δ 2.9-3.9ppm (-O-CH ３ , -CONH-CH ２ -CH ２ -), 2.3-2.9ppm (-CH ２ -CH(O-CH ３ )-), 2.0-2.3ppm (-CH ２ -N(CH ３ ) ２ ), 1.8-2.0ppm (-N(CH ３ ) ２ ), 1.0-1.5ppm (-CONH-CH ２ -CH ２ -CH ２ -CH ２ -), 1.3-4.0 ppm (polymer main chain) Molar ratio of repeating units: x / y = 75 / 25 ( １ H NMR) Yield of Polymer K: 6.2 g

[0224] The number-average molecular weight and polydispersity index (weight-average molecular weight / number-average molecular weight) of the obtained polymer K were determined by gel permeation chromatography using a differential refractometer as a detector. A JASCO PU-2080 pump, a JASCO RI-2031 detector (differential refractometer), and a UV-2075 detector (UV) were used. The column used consisted of two TSKGel G3000PWXL and TSKGel G5000PWXL columns (column size: 4.6 mm x 25 cm) connected together, both manufactured by Tosoh Corporation. The developing solvent was 0.1 M phosphate buffer adjusted to pH 7.4. The measurement conditions were a flow rate of 0.45 mL / min, a column temperature of 40°C, a sample concentration of 20 mg / mL, and an injection volume of 100 μL. Polyethylene glycol was used as a standard. As a result of gel permeation chromatography, the number average molecular weight of polymer K was 46,900 and its polydispersity index was 2.72.

[0225] Example 1 Preparation of Cryoprotectant A 2% by mass of polymer A, 2% by mass of proline (Tokyo Chemical Industry Co., Ltd.), 0.1% by mass of methylcellulose (Tokyo Chemical Industry Co., Ltd.), 10% by mass of dimethyl sulfoxide (hereinafter abbreviated as "DMSO") (Fujifilm Wako Pure Chemical Industries, Ltd.), 40% by mass of fetal bovine serum (hereinafter abbreviated as "FBS") (Biosera), 30% by mass of 25 mM phosphate buffer, and water (the remainder) were mixed to prepare cryoprotectant A. Note that the methylcellulose used was one whose viscosity at 20°C of an aqueous solution with a concentration of 2% by mass was 20 to 30 mPa s.

[0226] Examples 2 to 7: Preparation of cryoprotectants B to G Cryoprotectants B to G were prepared in the same manner as in Example 1, except that polymer A was replaced with one of polymers B to G.

[0227] Comparative Example 1 Preparation of Cryoprotectant H Cryoprotectant H was prepared in the same manner as in Example 1, except that proline and methylcellulose were not used.

[0228] Comparative Example 2 Preparation of Cryoprotectant I Cryoprotectant I was prepared in the same manner as in Example 1, except that polymer A and methylcellulose were not used.

[0229] Examples 8 and 9: Preparation of cryoprotectants J and K Cryoprotectants J and K were prepared in the same manner as in Example 1, except that polymer J or K was used instead of polymer A.

[0230] Example 10 Preparation of Cryoprotectant L 4% by mass of polymer A, 4% by mass of proline (Tokyo Chemical Industry Co., Ltd.), 0.2% by mass of methylcellulose (Tokyo Chemical Industry Co., Ltd.), 20% by mass of DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.), 50% by mass of FBS (Biosera), 20% by mass of 25 mM phosphate buffer, and water (the remainder) were mixed to prepare cryoprotectant L. Note that the methylcellulose used was one whose viscosity at 20°C of an aqueous solution with a concentration of 2% by mass was 20 to 30 mPa s.

[0231] The compositions of cryoprotectants A to L obtained in Examples 1 to 10 and Comparative Examples 1 and 2 are shown in Table 1 below.

[0232]

[0233] Comparative Example 3 Preparation of Cryoprotectant M Cryoprotectant M was prepared by mixing 10% by mass of DMSO, 10% by mass of FBS (Biosera), and the remainder of Dulbecco's modified Eagle's medium.

[0234] Comparative Example 4: Existing cryoprotectant Cellbanker 1 plus (manufactured by Zenogen Pharma) was used as an existing cryoprotectant.

[0235] Test Example 1: Cryopreservation test of two-dimensionally cultured cells 1 × 10 HepG2 cells were suspended in Dulbecco's modified Eagle's medium containing 10% by mass of FBS (hereinafter referred to as "medium 1"). ４ 100 μL of the solution was seeded into each well of a 96-well plate so that the cells were 100 μL per well. ２ Two-dimensional culture was performed in an incubator at 37°C for 72 hours. Thereafter, Medium 1 was removed from each well, and 50 μL of each cryoprotectant obtained in Examples 1 to 7 or Comparative Examples 1 or 2 was added to each well (1 × 10 cells to be frozen and preserved). ４ The amount of cryoprotectant used per cell: approximately 50 μL.

[0236] Cells on a 96-well plate containing a cryoprotectant were cooled at 1°C / min and cryopreserved at -80°C for 5 days. After cryopreservation, the 96-well plate was left at -20°C for 30 minutes, and then 50 μL of Medium 1 preheated to 37°C was added to each well. The plate was then left at 37°C for 5 minutes to thaw. After thawing, the cryoprotectant was removed by aspiration, and the cells in each well were washed once with Medium 1. 100 μL of Medium 1 was then added to each well. The cells were cultured for 48 hours, and the resulting cells were subjected to various assays. The results are shown in Table 2.

[0237] (Calculation of viability of 2D cultured cells) After cryopreservation and thawing according to the above procedure, 100 μL of Cell titerglobe 2.0 (Promega) was added to each well of a 96-well plate, and the plate was left to stand at room temperature for 10 minutes. The luminescence intensity (hereinafter referred to as "luminescence intensity after thawing") was then measured using a plate reader. The same procedure was also performed on HepG2 cells before cryopreservation, and the luminescence intensity (hereinafter referred to as "luminescence intensity before cryopreservation") was measured. Using the obtained luminescence intensities after thawing and before cryopreservation, the cell viability (%) was calculated using the following formula: Cell viability (%) = 100 × luminescence intensity after thawing / luminescence intensity before cryopreservation.

[0238] (Confirmation of morphology (cell detachment and morphological abnormalities) of two-dimensionally cultured cells) After cryopreservation and thawing according to the above procedure, each well on the 96-well plate was observed using an optical microscope for the presence or absence of cell detachment and morphological abnormalities of the cells. In addition, morphological abnormalities of the cells were evaluated according to the following criteria. Evaluation criteria ◎: No change in cell morphology after thawing compared to before cryopreservation ○: Some of the cells after thawing had a morphology that had changed to that of fibroblasts ×: Many of the cells after thawing had a morphology that had changed to that of fibroblasts

[0239] (Calculation of urea secretion rate of two-dimensionally cultured cells) After cryopreservation and thawing in the above procedure, medium 1 was removed from each well on the 96-well plate, and DMEM containing 1 mM ammonium chloride was then added to each well. ２ The cells were cultured in an incubator at 37°C for 24 hours, and the culture supernatant was collected. The amount of urea in the culture supernatant (hereinafter referred to as "urea amount after thawing") was measured using a Urea Assay Kit (manufactured by Biochain). The same procedure was also performed on HepG2 cells before freezing, and the amount of urea in the culture supernatant (hereinafter referred to as "urea amount before cryopreservation") was measured. The urea secretion rate (%) was calculated using the obtained amounts of urea after thawing and before cryopreservation according to the following formula: Urea secretion rate (%) = 100 × amount of urea after thawing / amount of urea before cryopreservation.

[0240]

[0241] As shown in Table 2, cryoprotectants A to G of Examples 1 to 7 maintained a high cell viability of two-dimensionally cultured cells compared to cryoprotectants H and I of Comparative Examples 1 and 2. Furthermore, by using cryoprotectants A to G, it is possible to suppress cell detachment and morphological abnormalities of two-dimensionally cultured cells and maintain cell function (urea secretion ability).

[0242] <Test Example 2: Cryopreservation test of 3D cultured cells> 4 × 10 HeG2 cells suspended in medium 1 ３ 100 μL of the culture medium was seeded into a low-adhesion 96-well U-bottom plate (EZ-BindShut® II, IWAKI) at 100 μL / well, centrifuged at 300×g for 5 minutes, and then incubated for 1 hour at 25°C for 1 hour. ２ Three-dimensional culture was performed in an incubator at 37°C for 72 hours. After culture, four of the resulting spheroids were transferred to one Eppendorf tube, the supernatant was removed to approximately 100 μL, and the spheroids were washed with 1000 μL of Medium 1. After washing, cryoprotectant B obtained in Example 2, cryoprotectant H obtained in Comparative Example 1, cryoprotectant I obtained in Comparative Example 2, or the existing cryoprotectant of Comparative Example 4 (900 μL) was added to the Eppendorf tube (1 × 10 cells to be frozen and stored). ４ The amount of cryoprotectant used per cell: approximately 560 μL.

[0243] The resulting mixture was transferred to a cryotube and placed in a BICELL (manufactured by Nippon Freezer Co., Ltd.), cooled at 1°C / min, and the spheroids were frozen and stored at -80°C for 5 days. The removed cryotube was then placed in a 37°C water bath for 2 minutes to thaw the spheroids. After thawing, the thawed spheroids were added to a 15 mL centrifuge tube containing 9 mL of medium 1, the supernatant was removed to about 1 mL, and the remainder was transferred to an Eppendorf tube. After that, the supernatant was removed to about 100 μL, and the spheroids were washed with 1 mL of medium 1, transferred to a low-adhesion 96-well U-bottom plate, and then incubated in CO ２ The cells were cultured in an incubator at 37°C for 48 hours, and the cultured spheroids were subjected to various assays. The results are shown in Table 3.

[0244] (Calculation of recovery rate of 3D cultured cells) The number of spheroids placed in the cryotube before cryopreservation (hereinafter referred to as "number of spheroids before cryopreservation") and the number of spheroids recovered from the cryotube after cryopreservation and thawing (hereinafter referred to as "number of spheroids after thawing") were measured by optical microscope observation, and the spheroid recovery rate (%) was calculated using the following formula: Spheroid recovery rate (%) = 100 × number of spheroids after thawing / number of spheroids before cryopreservation.

[0245] (Calculation of Viability of 3D-Cultured Cells) To the spheroids that had been cryopreserved and thawed and cultured for 48 hours according to the above procedure (hereinafter referred to as "spheroids 1 after thawing and culture"), 50 μL of CellTiter-Glo 3D (Promega) was added, and the luminescence intensity (hereinafter referred to as "luminescence intensity 1 after thawing and culture") was measured using a Microplate Reader Infinite M1000 PRO (TECAN). The same procedure was also performed on the spheroids before cryopreservation, and the luminescence intensity (hereinafter referred to as "luminescence intensity 1 before cryopreservation") was measured. Using the obtained luminescence intensities after thawing and culture and before cryopreservation, the cell viability (%) was calculated according to the following formula: Cell viability (%) = 100 × luminescence intensity 1 after thawing and culture / luminescence intensity 1 before cryopreservation.

[0246] (Confirmation of morphological changes in 3D cultured cells) The presence or absence of morphological changes in spheroids 1 after thawing and culturing was observed using an optical microscope and evaluated according to the following criteria. Evaluation criteria ◎: The morphology of spheroids 1 after thawing and culturing remains unchanged compared to that before cryopreservation ○: The morphology of some spheroids 1 after thawing and culturing is distorted ×: The spheroids 1 are fragmented after thawing and culturing

[0247] (Calculation of the percentage of dead cells in 3D cultured cells) Spheroid 1 after thawing and culture was stained with a LIVE / DEAD Viability / Cytotoxicity Kit (manufactured by Thermo Fisher Scientific) and observed with a confocal laser scanning confocal microscope LSM880 (manufactured by Zeiss). The percentage of dead cells (%) (= 100 × number of dead cells / total number of cells) was calculated and evaluated according to the following criteria. Evaluation criteria ◎: The percentage of dead cells in spheroid 1 after thawing and culture is less than 20%. ○: The percentage of dead cells in spheroid 1 after thawing and culture is 20 to 80%. ×: The percentage of dead cells in spheroid 1 after thawing and culture is more than 80%.

[0248]

[0249] As shown in Table 3, the use of cryoprotectant B in Example 2 improved the recovery rate of spheroids and cell viability, and suppressed morphological changes and dead cells.

[0250] <Test Example 3: Cryopreservation test of 3D cultured cells> 1 x 10 HeG2 cells suspended in medium 1 ５ 1000 μL of the solution was seeded into an EZSPHERE® SP 24-well microplate (manufactured by IWAKI) at 1000 cells / well, and then incubated with CO ２ Three-dimensional culture was performed in an incubator at 37°C for 72 hours. After culture, spheroids from 10 wells were transferred to a 15 mL centrifuge tube, the supernatant was removed to approximately 1000 μL, washed with 9000 μL of medium 1, and the culture supernatant was again removed to 1000 μL. Subsequently, 4000 μL of medium 1 was added to obtain 5000 μL of a spheroid mixture. Cryoprotectant A obtained in Example 1, cryoprotectant M obtained in Comparative Example 3, or the existing cryoprotectant (500 μL) of Comparative Example 4 was added to 50 μL of the spheroid mixture (1 × 10 cells to be frozen and stored). ４ The amount of cryoprotectant used per cell: approximately 500 μL.

[0251] The resulting mixture was transferred to a cryotube and placed in a BICELL (manufactured by Nippon Freezer Co., Ltd.), cooled at 1°C / min, and the spheroids were frozen and stored at -80°C for 5 days. The removed cryotube was then placed in a 37°C water bath for 2 minutes to thaw the spheroids. After thawing, the thawed spheroids were added to a 15 mL centrifuge tube containing 9 mL of medium 1, the supernatant was removed to about 1 mL, and the remainder was transferred to an Eppendorf tube. After that, the supernatant was removed to about 100 μL, and the spheroids were washed with 1 mL of medium 1, transferred to a low-adhesion 96-well flat-bottom plate, and then cooled to 5°C. ２ The cells were cultured in an incubator at 37°C for 24 hours, and the cultured spheroids were subjected to various assays. The results are shown in Table 4.

[0252] (Calculation of Viability of 3D-Cultured Cells) To the spheroids that had been cryopreserved and thawed and cultured for 24 hours according to the above procedure (hereinafter referred to as "spheroids 2 after thawing and culture"), 50 μL of CellTiter-Glo 3D (Promega) was added, and the luminescence intensity (hereinafter referred to as "luminescence intensity 2 after thawing and culture") was measured using a Microplate Reader Infinite M1000 PRO (TECAN). The same procedure was also performed on the spheroids before cryopreservation, and the luminescence intensity (hereinafter referred to as "luminescence intensity 2 before cryopreservation") was measured. Using the obtained luminescence intensities after thawing and culture and before cryopreservation, the cell viability (%) was calculated according to the following formula: Cell viability (%) = 100 × luminescence intensity 2 after thawing and culture / luminescence intensity 2 before cryopreservation.

[0253] (Calculation of the percentage of dead cells in 3D cultured cells) Spheroids 2 after thawing and culture were stained with a LIVE / DEAD Viability / Cytotoxicity Kit (manufactured by Thermo Fisher Scientific) and observed with a confocal laser scanning confocal microscope LSM880 (manufactured by Zeiss). The percentage of dead cells (%) (= 100 × number of dead cells / total number of cells) was calculated and evaluated according to the following criteria. Evaluation criteria ◎: The percentage of dead cells in spheroids 2 after thawing and culture is less than 20%. ○: The percentage of dead cells in spheroids 2 after thawing and culture is 20 to 80%. ×: The percentage of dead cells in spheroids 2 after thawing and culture is more than 80%.

[0254]

[0255] As shown in Table 4, the use of cryoprotectant A of Example 1 improved the cell viability of the spheroids and suppressed cell death.

[0256] Test Example 4: Cryopreservation test of cells suspended in medium HepG2 cells were detached from a polystyrene dish (manufactured by Corning) by treatment with trypsin-EDTA (manufactured by Sigma-Aldrich) at 37°C for 5 minutes and suspended in medium 1 (amount of cells per mL of medium: 1 x 10 ６ The resulting mixture was centrifuged at 300 × g for 5 minutes, and the supernatant was removed. The HepG2 cells from which the supernatant had been removed were mixed with cryoprotectant A obtained in Example 1, cryoprotectant M obtained in Comparative Example 3, or the existing cryoprotectant in Comparative Example 4 to prepare a mixture containing HepG2 cells and a cryoprotectant (1 × 10 cells to be cryopreserved). ４ Amount of cryoprotectant used per cell: approximately 10 μL, concentration of HepG2 cells in the mixture: 1 × 10 ６The resulting mixture was transferred in 1000 μL aliquots into cryotubes, placed in BICELL (manufactured by Nippon Freezer Co., Ltd.), cooled at 1°C / min, and cryopreserved at -80°C for 5 days. The removed cryotubes were then placed in a 37°C water bath for 2 minutes to thaw the cells. The thawed mixture was added to a 15 mL centrifuge tube containing 9 mL of Medium 1 and centrifuged at 300 × g for 5 minutes. The medium and cryoprotectant were then aspirated and the resulting cells were suspended in 10 mL of Medium 1. The cell suspension obtained by the above procedure was then subjected to various assays. The results are shown in Table 5.

[0257] (Calculation of recovery rate and viability of cells after thawing) 100 μL of Trypan Blue Solution, 0.4% (manufactured by Thermo Fisher Scientific) was added to 100 μL of each cell suspension that had been cryopreserved and thawed by the above procedure, and mixed. The cell number and cell viability of each cell suspension were calculated using a Countess 2 automatic cell counter (manufactured by Thermo Fisher Scientific). The obtained cell numbers were used according to the following formula: Cell recovery rate (%) = 100 × number of cells after thawing (1 × 10 ６ cells) / number of cells before cryopreservation (1 x 10 ６ The cell recovery rate (%) was calculated from the number of cells.

[0258]

[0259] As shown in Table 5, the use of cryoprotectant A of Example 1 improved the cell recovery rate and cell viability after thawing.

[0260] Test Example 5: Cryopreservation test of 2D cultured cells (without step B) 1 x 10 HeG2 cells suspended in medium 1 ４ 100 μL of the solution was seeded into each well of a 96-well plate so that the cells were 100 μL per well. ２ Two-dimensional culture was performed in an incubator at 37°C for 72 hours. Thereafter, without removing the medium 1 from each well, 100 µL of the cryoprotectant L obtained in Example 10 was added to each well (1 x 10 cells to be frozen and stored). ４The amount of cryoprotectant used per cell: approximately 100 μL.

[0261] Cells on a 96-well plate containing a cryoprotectant were cooled at 1°C / min and cryopreserved at -80°C for 5 days. After cryopreservation, the 96-well plate was left at -20°C for 30 minutes, and then 100 μL of medium 1 warmed to 37°C was added to each well. The plate was then left at 37°C for 5 minutes to thaw. After thawing, the cryoprotectant was removed by suction, and the cells in each well were washed once with medium 1. 100 μL of medium 1 was then added to each well. The cells were cultured for 48 hours, and the resulting cells were subjected to the assays "Calculation of the viability of 2D-cultured cells" and "Confirmation of the morphology (cell detachment and morphological abnormalities) of 2D-cultured cells" in Test Example 1. The results are shown in Table 6. Table 6 compares the results of Test Example 1, in which cryoprotectant A of Example 1 was used and step B (culture supernatant removal step) was performed, with the results of Test Example 5, in which cryoprotectant L of Example 10 was used and step B was not performed.

[0262]

[0263] As shown in Table 6, the cell viability of Test Example 1, in which cryoprotectant A of Example 1 was used and step B (culture supernatant removal step) was performed, and the cell viability of Test Example 5, in which cryoprotectant L of Example 10 was used and step B was not performed, were both 100% or higher. Furthermore, both Test Example 1, in which step B was performed, and Test Example 5, in which step B was not performed, were able to suppress cell detachment and morphological abnormalities in two-dimensionally cultured cells.

[0264] <Test Example 6: Cryopreservation test of two-dimensionally cultured mesenchymal stem cells> 10% by mass FBS and 2 × 10 －７ 2.5 × 10 human subcutaneous fat-derived mesenchymal stem cells suspended in α-MEM medium containing 10% by mass of bFGF (hereinafter referred to as "medium 2") were cultured. ３ 100 μL of the solution was seeded into each well of a 96-well plate so that the cells were 100 μL per well. ２ Two-dimensional culture was performed in an incubator at 37°C for 72 hours. Thereafter, Medium 2 was removed from each well, and 50 µL of each cryoprotectant obtained in Examples 1, 2, 8, or 9, or Comparative Example 3 was added to each well (1 x 10 cells to be frozen and stored).４ The amount of cryoprotectant used per cell: approximately 200 μL.

[0265] Cells on a 96-well plate containing a cryoprotectant were cooled at 1°C / min and cryopreserved at -80°C for 5 days. After cryopreservation, the 96-well plate was left at -20°C for 30 minutes, and then 50 μL of Medium 2 preheated to 37°C was added to each well. The plate was then left at 37°C for 5 minutes to thaw. After thawing, the cryoprotectant was removed by aspiration, and 100 μL of Medium 2 was added to each well. The cells were cultured for 48 hours, and the resulting cells were subjected to various assays. The results are shown in Table 7.

[0266] (Calculation of viability of 2D-cultured mesenchymal stem cells) After cryopreservation and thawing according to the above procedure, 100 μL of Cell titerglob 2.0 (Promega) was added to each well of a 96-well plate, and the plate was left to stand at room temperature for 10 minutes. The luminescence intensity (hereinafter referred to as "luminescence intensity after thawing") was then measured using a plate reader. The same procedure was also performed on human subcutaneous adipose-derived mesenchymal stem cells before cryopreservation, and the luminescence intensity (hereinafter referred to as "luminescence intensity before cryopreservation") was measured. Using the obtained luminescence intensities after thawing and before cryopreservation, the cell viability (%) was calculated using the following formula: Cell viability (%) = 100 × luminescence intensity after thawing / luminescence intensity before cryopreservation.

[0267]

[0268] As shown in Table 7, cryoprotectants A, B, J, and K of Examples 1, 2, 8, and 9 all maintained high cell viability of 2D-cultured mesenchymal stem cells compared to cryoprotectant M of Comparative Example 3.

[0269] The cryoprotective agents of the present invention are useful for protecting cells (eg, two-dimensionally cultured cells, three-dimensionally cultured cells, and cells suspended in a medium) during cryopreservation.

[0270] This application is based on Japanese Patent Application No. 2023-174728, the contents of which are incorporated in their entirety herein.

Claims

1. A cryoprotectant for cells comprising an amphoteric polymer, a hydrophobic amino acid, and water-soluble cellulose, wherein the amphoteric polymer is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , *-N ＋ (CH ３ ) ２ -*, *-S ＋ (CH ３ ) ２ , and *-S ＋ (CH ３ a cationic group selected from the group consisting of *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-O-P(O)(O － and an anionic group selected from the group consisting of --O--* (wherein * represents a bonding position).

2. The amphoteric polymer has the formula (1): (In formula (1), * represents a bonding position, R １ and R ２ each independently represents a hydrogen atom or a methyl group; ３ represents a methylene group; ４ O represents an oxyalkylene group having 2 to 4 carbon atoms, and n represents a number from 0 to 100.) and a repeating structure A represented by formula (2): In formula (2), * represents a bonding position, R ３ represents a hydrogen atom or a methyl group; １ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), p represents an integer of 1 to 6, and X １ is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position) and a repeating structure B represented by formula (3): In formula (3), * represents a bonding position, R ４ represents a hydrogen atom or a methyl group; ２ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), q represents an integer of 0 to 6, and Y １ is *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S (O) ２ (O － ) (wherein * represents a bond position, and C ６ H ４ 3. The cryoprotectant according to claim 1, having a repeating structure C represented by the formula: and a molar ratio of the repeating structure B to the sum of the repeating structure B and the repeating structure C is 0.1 or more and 0.9 or less.

3. In the formula (1), R １ is a hydrogen atom, R ２ is a methyl group, A ３ is absent or is a methylene group, ４ O is an oxyethylene group, and n is a number from 0 to 50; ３ is a hydrogen atom, and A １ is a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), p is an integer of 2 to 4, and X １ *-N ＋ (CH ３ ) ２ H and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position), and in formula (3), R ４ is a hydrogen atom, and A ２ is absent, q is 0, and Y １ *-CO-O － The cryoprotectant according to claim 2, wherein * represents a binding site.

4. The cryoprotectant of claim 3, wherein p is 2.

5. The amphoteric polymer has the formula (4): In formula (4), * represents a bonding position, R ５ represents a hydrogen atom or a methyl group; ５ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), r represents an integer of 1 to 6, and X ２ is *-N ＋ H ３ , *-N ＋ (CH ３ ) H ２ , *-N ＋ (CH ３ ) ２ H, *-N ＋ (CH ３ ) ３ , and *-S ＋ (CH ３ ) ２ (wherein * represents a bonding position) and a repeating structure D represented by formula (5): In formula (5), * represents a bonding position, R ６ represents a hydrogen atom or a methyl group; ６ represents a group selected from the group consisting of *-CO-O-* and *-CO-NH-* (wherein * represents a bonding position), s represents an integer of 0 to 6, and Y ２ is *-CO-O － , *-S(O) ２ (O － ), *-OP(O)(OH)(O － ), and *-C ６ H ４ -S (O) ２ (O － ) (wherein * represents a bond position, and C ６ H ４ represents a phenylene group.) represents a group selected from the group consisting of: and a repeating structure E represented by: wherein the molar ratio of the repeating structure D to the sum of the repeating structure D and the repeating structure E is 0.1 or more and 0.9 or less.

6. In the formula (4), R ５ is a methyl group, A ５ is *--CO-O--* (wherein * represents a bond position), r is 2, and X ２ *-N ＋ (CH ３ ) ２ H (in the formula, * represents a bonding position), and ６ is a methyl group, A ６ is absent or is *--CO-O--* (wherein * represents a bonding position), s is an integer of 0 to 3, and Y ２ *-CO-O － Or *-S(O) ２ (O － 6. The cryoprotectant according to claim 5, wherein * represents a binding site.

7. The amphoteric polymer has the formula (6): (In formula (6), * represents a bonding position.) or a group represented by formula (7): The cryoprotectant according to claim 1 , which has a group represented by the following formula (7): (in formula (7), * represents a bonding position).

8. The cryoprotectant according to any one of claims 1 to 7, wherein the number average molecular weight of the amphoteric polymer is 1,000 to 2,000,000.

9. The cryoprotectant according to any one of claims 1 to 7, wherein the hydrophobic amino acid is proline.

10. A cryoprotectant according to any one of claims 1 to 7, wherein the water-soluble cellulose is at least one selected from the group consisting of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

11. A method for cryopreserving cells, comprising: step 1 of mixing cells with the cryoprotectant according to any one of claims 1 to 7; and step 2 of freezing the mixture containing the cells and the cryoprotectant.

12. The method according to claim 11, wherein the cells are two-dimensionally cultured cells, and the method comprises, prior to step 1, step A of two-dimensionally culturing adherent cells in a medium on a culture substrate, step 1 being step C of mixing the two-dimensionally cultured cells with a cryoprotectant according to any one of claims 1 to 7, and step 2 being step D of freezing the mixture comprising the two-dimensionally cultured cells on the culture substrate and the cryoprotectant.

13. The method according to claim 12, further comprising a step B between steps A and C of removing the culture supernatant from the two-dimensionally cultured cells.

14. The method according to claim 11, wherein the cells are three-dimensionally cultured cells, and the method comprises, prior to step 1, a step E of three-dimensionally culturing the cells in a medium, the step 1 being a step G of mixing the three-dimensionally cultured cells with a cryoprotectant according to any one of claims 1 to 7, and the step 2 being a step H of freezing the mixture comprising the three-dimensionally cultured cells and the cryoprotectant.

15. The method according to claim 14, further comprising a step F between steps E and G, of transferring the three-dimensionally cultured cells into a cryopreservation container and removing the culture supernatant.

16. The method according to claim 11, wherein the method for cryopreserving cells is a method for cryopreserving cells suspended in a medium, and includes, prior to step 1, step I of bringing the cells into a state of being suspended in a medium, step 1 being step K, and step 2 being step L.

17. The method of claim 16, further comprising, between steps I and K, a step J of removing the supernatant from the mixture comprising the medium and the cells.

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