Cryoprotective solution and cryopreservation method

Abstract

To provide a cryoprotective solution characterized by high dispersibility of magnetic nanoparticles and a superior cryoprotective effect for biological samples.SOLUTION: A cryoprotective solution for biological samples comprises a copolymer having a hydrophilic structural unit represented by formula (1) and a hydrophobic structural unit represented by formula (2) [where R1 and R5 are H, a methyl group, or the like; R2 to R4 are hydrocarbon groups having 1-10 carbon atoms; L1 and L2 are a single bond, an ether bond, etc.; R6 is a hydrocarbon group having 1 or more carbon atoms].SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cryoprotectant and a cryopreservation method. [Background technology]

[0002] Tissue and organ transplants save the lives of many patients every year. However, there is a global shortage of tissues and organs for transplantation, which is one of the biggest challenges in the field of biomedicine. The period during which organs can be refrigerated is, for example, four hours for the heart and lungs, eight hours for the liver and pancreas, and 36 hours for the kidneys, and in many cases, they are discarded before they can be transplanted. For this reason, there is a demand for the development of technology to stably preserve donated tissues and organs for long periods of time.

[0003] One method for long-term storage of biological samples is to freeze and store them at subzero temperatures. However, when cells are frozen and thawed, ice crystals are formed inside and outside the cells, which can physically damage the cell membrane. For this reason, most cells usually die after a cycle of freezing and thawing. Therefore, in order to freeze and store biological samples in a usable state after thawing, it is important to freeze and thaw them quickly. However, there has been no established technology for thawing biological samples by heating them quickly and uniformly in a manner that does not damage cells or tissues, especially when the biological sample contains a large amount of cells, thick tissues, or organs.

[0004] The inventors have reported that by using a "nanowarming" technology that utilizes the principle that magnetic nanoparticles generate heat in an alternating magnetic field, it is possible to uniformly and rapidly heat the cryoprotectant solution containing the biological sample, regardless of its volume, and that this makes it possible to thaw the frozen cell solution without inducing ice crystal formation due to recrystallization caused by a slow heating rate (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Bioeng Transl Med. 2023;8(4):e10416 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, the inventors have succeeded in uniformly and rapidly warming biological tissue by perfusing a cryoprotectant containing magnetic nanoparticles. However, the inventors have also found that when using this technology, the magnetic nanoparticles are poorly dispersed in the cryoprotectant, and the magnetic nanoparticles precipitate in the tissue after perfusion and cannot be washed away.

[0007] The present invention has been made in consideration of the current state of the prior art described above, and its main object is to provide a cryoprotective liquid that has high dispersibility for magnetic nanoparticles and excellent cryoprotective effect for biological samples. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to achieve the above-mentioned object. As a result, they have found that by adding a copolymer having both a predetermined hydrophilic constituent unit and a hydrophobic constituent unit to a cryoprotective solution, it is possible to easily provide a cryoprotective solution that has high dispersibility of magnetic nanoparticles and excellent cryoprotective effect for biological samples. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention includes the following configurations.

[0009] Item 1. A cryoprotectant for biological samples, General formula (1):

[0010] [ka] [In the formula, R 1 R represents a hydrogen atom, a methyl group, or an ethyl group. 2 , R 3 and R 4are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms. L1 represents a single bond, an ether bond, an ester bond, or an amide bond. p and q are the same or different and each represents an integer of 1 to 6. A hydrophilic structural unit represented by the formula: General formula (2):

[0011] [ka] [In the formula, R 5 R represents a hydrogen atom, a methyl group, or an ethyl group. 6 represents a hydrocarbon group having one or more carbon atoms. L2 represents a single bond, an ether bond, an ester bond, or an amide bond. A hydrophobic structural unit represented by the formula: The copolymer includes a copolymer having the formula: Cryoprotectant.

[0012] Item 2. The total amount of the structural units constituting the copolymer is 100 mol%, The content of the hydrophilic structural unit is 20 to 99 mol %, and The content of the hydrophobic structural unit is 1 to 80 mol %. Item 1. A cryoprotectant solution according to item 1.

[0013] Section 3. Said R 1 is a hydrogen atom or a methyl group, and R 5 is a hydrogen atom or a methyl group; Item 3. The cryoprotectant solution according to item 1 or 2.

[0014] Section 4. Said R 2 , R 3 and R 4 Item 4. The cryoprotectant solution according to any one of Items 1 to 3, wherein is a methyl group.

[0015] Item 5. The cryoprotectant according to any one of Items 1 to 4, wherein p and q are the same or different and are integers of 1 to 3.

[0016] Section 6. Said R 66. The cryoprotectant according to any one of items 1 to 5, wherein is a hydrocarbon group having two or more carbon atoms.

[0017] Item 7. The cryoprotectant according to any one of Items 1 to 6, wherein L1 and L2 are ester bonds.

[0018] Item 8. The cryoprotective solution according to any one of Items 1 to 7, wherein the content of the copolymer is 10% by mass or more, with the total amount of the cryoprotective solution being 100% by mass.

[0019] Item 9. Further containing dimethyl sulfoxide, formamide and propylene glycol, The content of the copolymer relative to the total amount of the cryoprotectant solution is A (w / w%); When the total content of the dimethyl sulfoxide, the formamide and the propylene glycol with respect to the total amount of the cryoprotective solution is B (w / v%), 55≦A+B, Item 9. The cryoprotectant solution according to any one of items 1 to 8.

[0020] Item 10. The cryoprotective solution according to any one of Items 1 to 9, wherein the biological sample is a cell, a tissue, or an organ.

[0021] Item 11. The cryoprotective solution according to Item 10, wherein the biological sample is hepatic parenchymal cells, liver tissue, or liver.

[0022] Item 12. The cryoprotectant solution according to any one of Items 1 to 11, comprising magnetic nanoparticles.

[0023] Item 13. A method for cryopreserving a biological sample, comprising a step of freezing the cryoprotective solution according to any one of items 1 to 12 and a biological sample. Effect of the Invention

[0024] According to the present invention, it is possible to provide a cryoprotective solution that has high dispersibility for magnetic nanoparticles and has an excellent cryoprotective effect for biological samples. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows the results of Examples 1 and 2, and is a graph showing the cell viability of mouse pancreatic β cells (MIN6) after freezing and thawing (from left to right: Reference Example 1, Example 1-1, Example 2-1, Comparative Example 1-1, Example 1-2, Example 2-2, Comparative Example 1-2, Example 1-3, Example 2-3, Comparative Example 1-3, Example 1-4, Example 2-4). [Diagram 2] FIG. 1 shows the results of Examples 3 to 5 and is a graph showing the cell viability of mouse pancreatic β cells (MIN6) after freezing and thawing (from left to right: unfrozen group (NF), EuroCollins buffer (EC), Reference Example 1, Comparative Example 1-1, Example 3-1, Example 4-1, Example 5-1, Comparative Example 1-2, Example 3-2, Example 4-2, Example 5-2, Comparative Example 1-3, Example 3-3, Example 4-3, Example 5-3, Comparative Example 1-4, Example 3-4, Example 4-4, Example 5-4, Comparative Example 1-5, Example 3-5, Example 4-5, Example 5-5). [Diagram 3] FIG. 1 shows the results of Examples 3 to 5 and is a graph showing the cell viability of primary rat hepatic parenchymal cells after freezing and thawing (from left to right: unfrozen group (NF), EuroCollins buffer (EC), Reference Example 1, Comparative Example 1-1, Example 3-1, Example 4-1, Example 5-1, Comparative Example 1-2, Example 3-2, Example 4-2, Example 5-2, Comparative Example 1-3, Example 3-3, Example 4-3, Example 5-3, Comparative Example 1-4, Example 3-4, Example 4-4, Example 5-4, Comparative Example 1-5, Example 3-5, Example 4-5, Example 5-5). [Figure 4] The photographs were taken of dispersions obtained by dispersing magnetite nanoparticles (5.0 mg / mL) in cryoprotectant solutions (VS55 (Reference Example 1), VS35 (Comparative Example 1-4), VS35 + 20 wt% PMB (Example 5-4) from the top) after a certain period of time (0 minutes, 30 minutes, and 60 minutes from the left). [Diagram 5] The photographs were taken immediately after immersion in liquid nitrogen of the cryoprotectants of Examples 3 to 5. On the left side of the table, photographs of an example of unsuccessful vitrification (EuroCollins buffer solution) and an example of successful vitrification (VS55; Reference Example 1) are shown. [Figure 6]1 is a graph showing the results of Comparative Example 2 (left: MIN6 cells, right: hepatic parenchymal cells). [Figure 7] 1 is a graph showing the results of Example 6 (from the left, unfrozen group, PMB (Example 5-4), PMPC (low molecular weight), PMPC, PCB). [Figure 8] 1 is a graph showing the results of Example 7 (from the left: unfrozen group, PMB37 (Production Example 7), PMB55 (Production Example 8), PMB73 (Production Example 9), PMPC (Production Example 10)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In this specification, the term "comprise" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0027] In this specification, the expression of a numerical range "A to B" means "greater than or equal to A and less than or equal to B."

[0028] As used herein, the term "cryoprotective solution" refers to a solution that, when frozen and stored together with a biological sample such as a cell, tissue, or organ, can reduce damage that may be caused to the sample by freezing and thawing.

[0029] 1. Cryoprotectant The cryoprotective solution of the present invention is a cryoprotective solution for a biological sample, General formula (1):

[0030] [ka] [In the formula, R 1 R represents a hydrogen atom, a methyl group, or an ethyl group. 2 , R 3 and R 4are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms. L1 represents a single bond, an ether bond, an ester bond, or an amide bond. p and q are the same or different and each represents an integer of 1 to 6. A hydrophilic structural unit represented by the formula: General formula (2):

[0031] [ka] [In the formula, R 5 R represents a hydrogen atom, a methyl group, or an ethyl group. 6 are the same or different and represent a hydrocarbon group having 1 to 10 carbon atoms. L2 represents a single bond, an ether bond, an ester bond, or an amide bond. A hydrophobic structural unit represented by the formula: By virtue of having such characteristics, the cryoprotective solution of the present invention can have high dispersibility for magnetic nanoparticles and excellent cryoprotective effect for biological samples.

[0032] The content of the copolymer is not particularly limited, but from the viewpoint of the cryoprotective effect and the dispersibility of the magnetic nanoparticles, it is preferably 10% by mass or more, more preferably 15 to 70% by mass, and even more preferably 20 to 50% by mass, of the total amount of the cryoprotective liquid being 100% by mass.

[0033] (1-1) Copolymer The copolymer used in the present invention has the above hydrophilic structural unit and the above hydrophobic structural unit. The cryoprotective solution of the present invention contains such an amphiphilic copolymer, so that the dispersibility of magnetic nanoparticles is high and the cryoprotective effect of biological samples can be excellent. When a hydrophilic polymer consisting of only hydrophilic structural units is used as a cryoprotective agent, the cell viability after freezing and thawing is usually significantly reduced. In contrast, the copolymer used in the present invention can penetrate into living cells by combining hydrophobic structural units, thereby avoiding physical damage to the cell membrane caused by the formation of ice crystals.

[0034] The content of the hydrophilic structural unit is not particularly limited, and a wide range of content can be widely adopted. Among them, the content of the hydrophilic structural unit is preferably 20 to 99 mol%, more preferably 25 to 75 mol%, and even more preferably 30 to 70 mol%, based on the total amount of the structural units constituting the copolymer as 100 mol%, from the viewpoint of the cryoprotection effect and the dispersibility of the magnetic nanoparticles.

[0035] The content of the hydrophobic structural unit is not particularly limited, and a wide range of content can be widely adopted. Among them, the content of the hydrophobic structural unit is preferably 1 to 80 mol%, more preferably 25 to 75 mol%, and even more preferably 30 to 70 mol%, based on the total amount of the structural units constituting the copolymer as 100 mol%, from the viewpoint of the cryoprotection effect and the dispersibility of the magnetic nanoparticles.

[0036] The copolymer may further have a structural unit other than the hydrophilic structural unit and the hydrophobic structural unit described below, so long as the effect of the present invention is not significantly impaired. As the other structural unit, a structural unit contained in a known polymer such as MPC polymer, vinyl polymer, poly(meth)acrylic acid, polyacrylic acid ester, polyacrylamide, etc. can be appropriately used.

[0037] The copolymer arrangement is not particularly limited, and any of random copolymers, alternating copolymers, block copolymers, graft copolymers, etc. can be used. Among them, the copolymer arrangement is preferably a random copolymer from the viewpoint of the cryoprotection effect and the dispersibility of the magnetic nanoparticles.

[0038] The ends of the copolymer are not particularly limited and may be either the hydrophilic structural unit or the hydrophobic structural unit.

[0039] The degree of polymerization of the copolymer is not particularly limited, but is preferably from 10 to 10,000, more preferably from 20 to 1,000, and even more preferably from 50 to 500, from the viewpoints of the freeze protection effect and the dispersibility of the magnetic nanoparticles.

[0040] The number average molecular weight of the copolymer is not particularly limited, but from the viewpoint of the cryoprotection effect and the dispersibility of the magnetic nanoparticles, it is preferably 1,000 to 1,000,000, more preferably 5,000 to 300,000, and even more preferably 5,000 to 100,000. The number average molecular weight is determined by gel permeation chromatography.

[0041] The weight average molecular weight of the copolymer is not particularly limited, but from the viewpoint of the cryoprotection effect and the dispersibility of the magnetic nanoparticles, it is preferably 1,000 to 1,000,000, more preferably 10,000 to 500,000, and even more preferably 10,000 to 100,000. The weight average molecular weight is determined by gel permeation chromatography.

[0042] The polydispersity of the copolymer is not particularly limited, but from the viewpoint of quality control, it is preferably 1 to 3, more preferably 1 to 2. The polydispersity is determined by gel filtration chromatography.

[0043] The glass transition temperature of the copolymer is not particularly limited, but from the viewpoint of the cryoprotection effect, it is preferably −196° C. to 0° C., more preferably −180° C. to −20° C., and even more preferably −150° C. to −80° C. The glass transition temperature is determined by differential scanning calorimetry or the like.

[0044] (1-2) Hydrophilic structural unit The copolymer used in the present invention has the general formula (1):

[0045] [ka] [In the formula, R 1 R represents a hydrogen atom, a methyl group, or an ethyl group. 2 , R 3 and R 4 are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms. L1 represents a single bond, an ether bond, an ester bond, or an amide bond. p and q are the same or different and each represents an integer of 1 to 6. The cryoprotective solution of the present invention has a hydrophilic structural unit represented by the formula: By selecting a specific hydrophilic structural unit, the cryoprotective solution of the present invention can have high dispersibility of magnetic nanoparticles and excellent cryoprotective effect for biological samples. If the hydrophilic structural unit does not contain the above unit, the solubility in water and the cryoprotective effect for cells can be significantly reduced.

[0046] In the general formula (1), R 1 R represents a hydrogen atom, a methyl group, or an ethyl group. 1 From the viewpoints of the cryoprotective effect and the dispersibility of the magnetic nanoparticles, a hydrogen atom or a methyl group is preferable, and a methyl group is more preferable.

[0047] In the general formula (1), R 2 , R 3 and R 4 are the same or different and represent a hydrocarbon group having 1 to 10 carbon atoms. The hydrocarbon group is not particularly limited, and examples thereof include linear alkyl groups having 1 to 10 carbon atoms (particularly 1 to 6), such as n-propyl, n-butyl, and n-pentyl groups; branched alkyl groups having 3 to 10 carbon atoms (particularly 3 to 6), such as isopropyl, sec-butyl, and tert-butyl groups; cyclic alkyl groups having 3 to 10 carbon atoms (particularly 5 to 8), such as cyclopentyl and cyclohexyl groups; aryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as phenyl and naphthyl groups; and heteroaryl groups having 6 to 10 carbon atoms (particularly 6 to 8), such as imidazolyl, pyridyl, and indolyl groups. Among them, R 2 , R 3 and R 4 From the viewpoint of the cryoprotective effect and dispersibility of the magnetic nanoparticles, these may be the same or different and are preferably a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, or a cyclic alkyl group having 3 to 6 carbon atoms, more preferably a linear alkyl group having 1 to 3 carbon atoms, and even more preferably a methyl group.

[0048] In general formula (1), L1 represents a single bond, an ether bond, an ester bond, or an amide bond. Among these, from the viewpoint of the cryoprotective effect and the dispersibility of the magnetic nanoparticles, L1 is preferably a single bond, an ester bond, or an amide bond, more preferably a single bond or an ester bond, and further preferably an ester bond.

[0049] In general formula (1), p and q are the same or different and are an integer of 1 to 6. From the viewpoint of the cryoprotective effect and dispersibility of the magnetic nanoparticles, p and q are the same or different and are preferably an integer of 1 to 3, more preferably 1 or 2, and even more preferably 2.

[0050] From the above, specific examples of hydrophilic structural units include, for example,

[0051] [ka] Examples of structural units include:

[0052] The hydrophilic structural unit is not particularly limited, but the side chain may or may not be free in the cryoprotectant solution. Examples of counter ions that the hydrophilic structural unit may have include H + , Na + , K + , O.H. - , Cl - , CO 3 2- , P.O. 4 3- and any cation or anion present in the cryoprotectant solution.

[0053] In the copolymer used in the present invention, the hydrophilic constitutional unit may be used alone or in combination of two or more kinds.

[0054] (1-3) Hydrophobic building blocks The copolymer used in the present invention has the general formula (2):

[0055] [ka] [In the formula, R 5 R represents a hydrogen atom, a methyl group, or an ethyl group. 6 represents a hydrocarbon group having one or more carbon atoms. L2 represents a single bond, an ether bond, an ester bond, or an amide bond. The compound has a hydrophobic structural unit represented by the formula:

[0056] In the general formula (2), R 5 represents a hydrogen atom, a methyl group, or an ethyl group. 5 From the viewpoints of the cryoprotective effect and the dispersibility of the magnetic nanoparticles, a hydrogen atom or a methyl group is preferable, and a methyl group is more preferable.

[0057] In the general formula (2), R 6 represents a hydrocarbon group having one or more carbon atoms. As the hydrocarbon group, R 2 , R 3 and R 4 Examples of the compounds are the same as those mentioned in the explanation of R 6 From the viewpoint of the cryoprotective effect and dispersibility of the magnetic nanoparticles, a hydrocarbon group having 2 or more carbon atoms is more preferable, a hydrocarbon group having 3 or more carbon atoms is more preferable, and a hydrocarbon group having 4 or more carbon atoms is even more preferable.

[0058] R 6 Some of the atoms in the hydrocarbon group represented by the formula (I) may be substituted with other atoms (e.g., fluoro). In this case, R 6 The hydrocarbon group represented by the formula (I) is preferably a fluoroalkyl group having one or more carbon atoms, and more preferably a perfluoroalkyl group having one or more carbon atoms.

[0059] In general formula (2), L2 represents a single bond, an ether bond, an ester bond, or an amide bond. Among these, from the viewpoint of the cryoprotective effect and the dispersibility of the magnetic nanoparticles, L2 is preferably a single bond, an ester bond, or an amide bond, more preferably a single bond or an ester bond, and further preferably an ester bond.

[0060] From the above, specific examples of hydrophobic structural units include, for example,

[0061] [ka] [In the formula, the definitions of each symbol are the same as above.] Examples of structural units include:

[0062] In the copolymer used in the present invention, the hydrophobic constitutional unit may be used alone or in combination of two or more kinds.

[0063] (1-4) Other ingredients The cryoprotectant of the present invention is preferably an aqueous solution of the copolymer, in which case the copolymer may be present in a dissolved state in a liquid medium containing water.

[0064] The liquid medium is not particularly limited as long as it is an aqueous solvent capable of dissolving the copolymer, and a wide variety of known aqueous solvents can be used. Among these, examples of the liquid medium include water and culture medium.

[0065] The water is not particularly limited, and examples thereof include purified water, pure water, and ion-exchanged water. In addition to these, examples of water as a liquid medium include known buffer solutions containing water. As the buffer solution, any buffer solution normally used in this field can be used, so long as it does not cause loss of physiological activity such as enzyme activity and antigenicity of proteins. Examples include EuroCollins buffer solution, phosphate buffer solution, Tris buffer solution, Good's buffer solution, glycine buffer solution, borate buffer solution, and the like, and these may be used in combination. As the buffer solution, EuroCollins buffer solution is preferred from the viewpoint of cryoprotection effect.

[0066] The medium is not particularly limited, and may be any known cell culture medium, such as Dulbecco's Modified Eagle's MEM medium (DMEM), α-MEM medium, Roswell Park Memorial Institute (RPMI) medium, F12 medium, TC199 medium, GMEM medium, etc.

[0067] The cryoprotective solution of the present invention may further contain components other than those described above, so long as the effects of the present invention are not significantly impaired. Examples of other components include known additives used in known cryoprotective solutions, such as cryoprotectants, sugars, proteins, salts, and surfactants.

[0068] Examples of cryopreservation agents include dimethyl sulfoxide (DMSO), formamide, glycerol, glycerin, ethylene glycol, propylene glycol, polyvinyl alcohol (PVA), sericin, betaine, polyethylene glycol (PEG), and the like.

[0069] Examples of sugars include saccharides such as lactose, sucrose, trehalose, and glucose, as well as polysaccharides such as alginic acid, pullulan, chitosan, dextran, and glucomannan.

[0070] Examples of proteins include fetal bovine serum (FBS), antifreeze peptides, bovine serum albumin, gelatin, and casein.

[0071] Examples of salts include amino acids such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, and histidine, and their salts; peptides such as glycylglycine; inorganic salts such as phosphates, borates, sulfates, and Tris salts; flavins, and organic acids such as acetic acid, citric acid, malic acid, maleic acid, and gluconic acid, and their salts.

[0072] Examples of the surfactant include polyoxyethylene alkyl ether, Tween 20, and Pluronic (registered trademark).

[0073] In addition, antibiotics may be added to the cryoprotective solution of the present invention.

[0074] Among them, the cryoprotective solution of the present invention is not particularly limited, but preferably contains dimethyl sulfoxide (DMSO), formamide (FD) and propylene glycol (PG) from the viewpoint of vitrification freezing. Moreover, the cryoprotective solution of the present invention preferably contains 55 g (55 w / v%) of these three components per 100 mL of the cryoprotective solution using EuroCollins buffer as the liquid medium, and a cryoprotective solution having such a composition (referred to as VS55) exhibits a high cryoprotective effect on cells.

[0075] When the cryoprotective solution of the present invention further contains dimethyl sulfoxide, formamide and propylene glycol, it is not particularly limited, but when the content of the copolymer is A (w / w%) relative to the total amount of the cryoprotective solution (100 w / w%), and the sum of the contents of the dimethyl sulfoxide, the formamide and the propylene glycol is B (w / v%) relative to the total amount of the cryoprotective solution (100 w / v%), from the viewpoint of the cryoprotective effect, it is preferable that 55≦A+B is satisfied, more preferably 55≦A+B≦80, and even more preferably 55≦A+B≦70. In this case, the sample is easily vitrified during freezing, and it is easy to avoid the formation of ice crystals that damage the biological sample.

[0076] On the other hand, from the viewpoint of maintaining the differentiation ability of stem cells contained in a biological sample, it is preferable that the cryoprotective solution of the present invention contains a small amount of DMSO, it is more preferable that it contains almost no DMSO, and it is even more preferable that it contains no DMSO at all.

[0077] The cryoprotective solution of the present invention is not particularly limited, but preferably contains magnetic nanoparticles from the viewpoint of enabling uniform rapid heating. Here, the magnetic nanoparticles preferably contain a compound containing one or more elements such as iron, and atoms that generate heat when placed in a magnetic field. In this case, even if the biological sample contains a large volume of cells, thick tissues, or organs, the entire sample can be thawed by uniformly and rapidly heating it, thereby making it possible to provide the cryopreserved sample in a usable state without damaging the cells or tissues.

[0078] There is no particular limitation on the magnetic nanoparticles as long as they are nanoparticles that exhibit superparamagnetism. From the viewpoint of enabling uniform and rapid heating, maghemite (γ-Fe 2 O 3 ) nanoparticles, magnetite (Fe 3 O 4 ) nanoparticles are preferred, and magnetite (Fe 3 O 4 ) nanoparticles are more preferred.

[0079] The particle size of the magnetic nanoparticles is usually 1000 nm or less, and from the viewpoint of enabling uniform and rapid heating, it is preferably 100 nm or less, more preferably 1 to 50 nm, and even more preferably 5 to 20 nm. The particle size is determined by observation with a transmission electron microscope.

[0080] The concentration of the magnetic nanoparticles is not particularly limited, but from the viewpoint of enabling uniform rapid heating, it is preferably 1 mg / mL or more, more preferably 3 to 50 mg / mL, and even more preferably 5 to 10 mg / mL.

[0081] Although there are no particular limitations, it is preferable that the magnetic nanoparticles are surface-modified to improve dispersibility in the cryoprotectant solution. Examples of such modifications include coating with silica, grafting with polyethylene glycol (PEG), and coating or grafting with phospholipid polymers.

[0082] There are no particular limitations on the magnetic nanoparticles, and for example, those described in Non-Patent Document 1 above can be used.

[0083] (1-5) Biological samples The cryoprotective solution of the present invention, having the above-mentioned characteristics, can improve the cryoprotective effect of biological samples, making it an excellent cryoprotective solution for biological samples.

[0084] The biological sample for which the cryoprotective solution of the present invention is used is not particularly limited as long as it is derived from a living organism, and can be widely applied to known biological samples. Among them, the biological sample is preferably a cell, tissue, or organ, from the viewpoint of the ease with which the copolymer used in the present invention can penetrate into living cells. In particular, it is usually difficult to freeze and preserve delicate cells such as hepatic parenchymal cells, liver tissue, or liver, but according to the cryoprotective solution of the present invention, even when the biological sample is hepatic parenchymal cells, liver tissue, or liver, these biological samples can be frozen and preserved in a state that can be used after thawing. As can be easily understood by those skilled in the art, the cryoprotective solution of the present invention is not limited to the above, and can also be used for cryopreservation of proteins, nucleic acids, etc. derived from a living organism that may be damaged by a freeze-thaw cycle.

[0085] The cells for which the cryoprotective solution of the present invention is used are not particularly limited, and a wide variety of known cells can be adopted, including, for example, stem cells such as sperm cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord blood cells, chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, nerve cells, vascular endothelial cells, smooth muscle cells, cardiac muscle cells, epithelial cells, hepatocytes, pancreatic β cells, kidney cells, blood cells, sperm, sperm cells, eggs, egg cells, fertilized eggs, other established cells for culture, and plant cells, and may also include artificial cells.

[0086] Tissues for which the cryoprotective solution of the present invention can be used include heart valves, blood vessels, skin, bone, pancreatic islets, nerves, cornea, cartilage, skeletal muscles, large blood vessels such as the adrenal gland (e.g., the pulmonary artery), arteries, veins, lymph nodes, etc., and may also include cell aggregates such as cell sheets, colonies, and spheroids, as well as biological tissues artificially produced by tissue engineering.

[0087] Organs for which the cryoprotective solution of the present invention can be used include the heart, lungs, liver, kidneys, pancreas, intestines, eyeballs, ovaries, testes, placenta, thymus, and bones, and may also include organoids made of cells.

[0088] The volume of the biological sample is not particularly limited, but is usually 1,000 mL or less, and from the viewpoint of enabling uniform rapid heating, it is preferably 500 mL or less, more preferably 100 mL or less, and even more preferably 50 mL or less. According to the cryoprotectant of the present invention, even if the biological sample contains a large volume of cells, thick tissues, or organs, the cryoprotectant can provide the cryopreserved sample in a usable state without damaging the cells or tissues by uniformly and rapidly heating and thawing the entire sample.

[0089] (1-6) Manufacturing method The cryoprotectant solution of the present invention can be produced, for example, by dissolving the copolymer in the liquid medium, but is not limited thereto.

[0090] The liquid medium is not particularly limited as long as it can dissolve the copolymer, but from the viewpoint of the cryoprotective effect, it is preferable that the liquid medium is a known cryoprotective liquid. That is, the cryoprotective liquid of the present invention is preferably produced using a known cryoprotective liquid. The production method includes the following steps: (A) a method of preparing a known cryoprotective solution and then mixing the cryoprotective solution with the copolymer in a predetermined ratio; and (B) A method of preparing a known cryoprotectant solution by mixing a given amount of the copolymer with a given ratio of a known cryoprotectant. Either of the above may be used.

[0091] The cryoprotectant solution may be stored after production until use, or may be prepared immediately before use.

[0092] The copolymer used in the present invention can be produced, for example, by free radical polymerization using a polymerization initiator such as an azo-based initiator, or by living radical polymerization using a catalyst such as a transition metal complex, but the production method is not limited thereto. Alternatively, the copolymer used in the present invention may be a commercially available product.

[0093] The copolymer used in the present invention is, for example, a copolymer represented by the general formula (1'):

[0094] [ka] [In the formula, the definitions of each symbol are the same as above.] and a hydrophilic monomer represented by General formula (2'):

[0095] [ka] [In the formula, the definitions of each symbol are the same as above.] and a hydrophobic monomer represented by the following formula (I):

[0096] The copolymer used in the present invention can be produced, for example, by mixing the above-mentioned hydrophilic monomer and hydrophobic monomer in predetermined amounts with a polymerization solvent and a polymerization initiator, and polymerizing the mixture under stirring at a predetermined temperature for a predetermined time in an inert gas (argon, nitrogen, etc.) atmosphere. The produced copolymer can be recovered by precipitating it in a poor solvent.

[0097] The hydrophilic monomer represented by the general formula (1') can be understood to be a so-called 2-(metha or etha)acryloxyethyl phosphorylcholine monomer when L1 is an ester bond.

[0098] The hydrophobic monomer represented by general formula (2') can be understood to be a so-called vinyl monomer when L2 is a single bond, a so-called vinyl ether monomer when L2 is an ether bond, a so-called (metha- or etha-)acrylic acid ester monomer when L2 is an ester bond, and a so-called (metha- or etha-)acrylamide monomer when L2 is an amide bond.

[0099] In the copolymerization of the hydrophilic monomer and the hydrophobic monomer, other monomers can also be copolymerized as long as the effects of the present invention are not significantly impaired.

[0100] The amounts of the hydrophilic monomer and the hydrophobic monomer used are not particularly limited and can be appropriately selected depending on the molar fraction in the resulting copolymer.

[0101] The polymerization solvent is not particularly limited as long as it dissolves the monomer, and examples thereof include ether solvents such as dioxane and tetrahydrofuran; amide solvents such as N,N-dimethylformamide; sulfoxide solvents such as dimethyl sulfoxide; aromatic hydrocarbon solvents such as benzene and toluene; alcohol solvents such as methanol, ethanol, isopropyl alcohol, amyl alcohol, and hexanol; and water.

[0102] The polymerization initiator is not particularly limited, and examples thereof include azo-based initiators such as azobisisobutyronitrile, azobisdimethylvaleronitrile, and azobis(isobutyrate)dimethyl; and peroxide-based initiators such as hydrogen peroxide, benzoyl peroxide, di-tert-butyl peroxide, and dicumyl peroxide.

[0103] The poor solvent is not particularly limited, and examples thereof include hydrocarbon solvents such as pentane, hexane, heptane, octane, nonane, and decane; and ether solvents such as dimethyl ether, ethyl methyl ether, diethyl ether, and diphenyl ether.

[0104] In addition to the above components, the copolymerization reaction can also use known acids, bases, oxidizing agents, reducing agents, catalysts, ligands, and other additives that are used in the copolymerization of vinyl polymers, acrylic acid ester polymers, acrylamide polymers, and the like.

[0105] The polymerization temperature is not particularly limited and is usually from 20 to 100°C.

[0106] The polymerization time is not particularly limited and is usually 1 to 48 hours.

[0107] 2. Cryopreservation method The cryopreservation method of the present invention includes a step of freezing the above-mentioned cryoprotective solution and a biological sample. By including this step, the above-mentioned cryoprotective solution can exert its effect and can exhibit a high cryoprotective effect on the biological sample. In addition, when the cryoprotective solution contains magnetic nanoparticles, the magnetic nanoparticles can be dispersed well, and thus the nanoparticles perfused in the tissue or organ can be efficiently collected.

[0108] The cryoprotective solution and biological sample used in the method of the present invention can be understood from the above description of the cryoprotective solution of the present invention.

[0109] (2-1) Freezing process The method of the invention may comprise the step of contacting the biological sample with a cryoprotectant solution prior to freezing.

[0110] The contact can be carried out, for example, by dispersing (suspending) the biological sample in the cryoprotective solution. In this case, it is preferable that the biological sample immediately before contact with the cryoprotective solution is substantially the biological sample itself (e.g., cells, tissues, organs, etc.) and contains as little other components (e.g., solvents, culture media, blood, solutes contained therein, etc.) so that the cryoprotective solution is not diluted by contact.

[0111] The freezing step is not particularly limited, but from the viewpoint of the cryoprotective effect, it is preferable to rapidly freeze the cryoprotective solution and the biological sample. For example, the rapid freezing method may include a method of cooling the biological sample in a freezer when the volume of the biological sample is small, or a method of rapidly freezing the entire biological sample of a large volume by immersing the cryoprotective solution containing the biological sample in liquid nitrogen.

[0112] The cooling rate in the freezing step is not particularly limited, but is usually 1° C. / min or more, and preferably 4° C. / min or more. When the cooling rate is equal to or higher than the critical cooling rate, it is easy to avoid the formation of ice crystals in the cryoprotectant solution.

[0113] The cooling is not particularly limited as long as it is possible to freeze the biological sample, and the temperature of the biological sample is preferably set to -40°C or lower, more preferably to -80°C or lower, and even more preferably to -120°C or lower.

[0114] There are no particular limitations on the method of storage of the frozen biological sample and cryoprotectant, and they may be stored, for example, in an ultra-low temperature freezer (about -80°C) or in a liquid nitrogen tank (about -196°C).

[0115] There is no particular limitation on the period of storage during freezing. According to the method of the present invention, a biological sample can be frozen and stored while maintaining its activity and function for, for example, 1 hour or more, preferably 24 hours or more, more preferably 1 week or more, and even more preferably 6 months or more.

[0116] (2-2) Thawing process The method of the present invention may comprise the step of thawing the frozen cryoprotectant and biological sample.

[0117] The thawing step is not particularly limited, but from the viewpoint of the cryoprotective effect, it is preferable to rapidly thaw the cryoprotective solution and the biological sample. Examples of rapid thawing methods include a method of warming the biological sample in a 37°C water bath when the volume of the biological sample is small, and a method of uniformly warming the entire large volume biological sample by "nanowarming", which causes magnetic nanoparticles dispersed in the cryoprotective solution to generate heat in an alternating magnetic field.

[0118] The heating rate in the thawing step is not particularly limited, but is usually 20° C. / min or more, and from the viewpoint of the cryoprotection effect, is preferably 30 to 200° C. / min, more preferably 40 to 150° C. / min, and even more preferably 50 to 100° C. / min. When the heating rate is equal to or higher than the critical heating rate, it is easy to avoid the formation of ice crystals in the cryoprotectant solution.

[0119] During thawing, the temperature difference between the surface temperature and the internal temperature of the biological sample is not particularly limited, but is usually 100°C or less, and from the viewpoint of the cryoprotection effect, it is preferably 10°C or less, more preferably 5°C or less, and even more preferably 1°C or less.

[0120] The heating is not particularly limited as long as it allows thawing, and the temperature of the biological sample is preferably -30°C or higher, more preferably 0°C or higher, more preferably 10°C or higher, and even more preferably 20°C or lower.

[0121] Nanowarming is not particularly limited, and can be carried out, for example, by the method described in the above-mentioned Non-Patent Document 1. Specifically, nanowarming can be carried out by applying an external AC magnetic field to a cryoprotectant solution containing magnetic nanoparticles using a high-frequency heating device.

[0122] The frequency of the alternating magnetic field is not particularly limited, but from the viewpoint of specific heating of a living body, it is preferably 1000 kHz or less, more preferably 50 to 500 kHz, and even more preferably 100 to 150 kHz.

[0123] The magnetic field strength of the alternating magnetic field is not particularly limited, but from the viewpoint of side effects on biological samples, it is preferably 100 kA / m or less, more preferably 1 to 60 kA / m, and even more preferably 10 to 40 kA / m.

[0124] The application time (irradiation time) of the AC magnetic field is not particularly limited, but from the viewpoint of side effects on the biological sample, it is preferably less than 1800 seconds, more preferably 1 to 1200 seconds, and even more preferably 10 to 300 seconds.

[0125] According to the method of the present invention, the activity and function of the thawed biological sample and the cells contained therein can be maintained at 50% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more, of those before freezing, but is not limited thereto. The method for evaluating the activity and function can be appropriately selected depending on the biological sample used and the cells contained therein. EXAMPLES

[0126] EXAMPLES Hereinafter, examples and comparative examples will be shown to more clearly illustrate the features of the present invention, but the present invention is not limited to the following examples.

[0127] In order to simplify the description of the specification, the following abbreviations may be used in the examples. Abbreviations: NF: unfrozen group, EC: EuroCollins buffer, DMSO dimethyl sulfoxide, FD: formamide, PG: propylene glycol, MPC: 2-methacryloyloxyethyl phosphorylcholine, BMA: butyl methacrylate, AIBN: azobisisobutyronitrile, SBMA: (2-(methacryloyloxy)ethyl)dimethyl-(3-sulfopropyl)ammonium, CBMA: 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, PEGMA: poly(ethylene glycol) methacrylate, EBiB: 2-ethyl bromoisobutyrate, TPMA: tris(2-pyridylmethyl)amine.

[0128] The properties of the synthesized copolymer were evaluated by the following tests. (Gel Permeation Chromatography) The molecular weight and polydispersity of the produced copolymer were determined by gel permeation chromatography using a differential refractometer as a detector. A PU-4180 (JASCO Corporation) was used as a pump, a RI-4030 (JASCO Corporation) was used as a detector (differential refractometer), and an OHpak SB-804 HQ or OHpak SB-805 HQ (Shoko Science Co., Ltd.) was used as a column. A mixed solvent of methanol:water = 7:3 (v / v%) containing 10 mM lithium bromide was used as a developing solvent. The flow rate was 0.5 mL / min. Polyethylene glycol was used as a standard substance.

[0129] [Manufacturing example 1: PMB37] To prepare a polymerization solution of Poly(MPC-co-BMA) (PMB), MPC (0.300 mol / L), BMA (0.70 mol / L), and AIBN (0.010 mol / L) were added to ethanol. The polymerization solution was bubbled with argon gas for 15 minutes, sealed, and then polymerized in an oil bath at 60°C for 20 hours. The polymerization solution was reprecipitated in a mixed solution of diethyl ether:chloroform = 9:1 (v / v%). The white precipitate was collected by decantation and dissolved in pure water. The solution was filtered through a filter with a pore size of 0.45 μm, and dialyzed for 3 days with a 3.5 kD dialysis membrane. The solution was freeze-dried to obtain a white solid PMB (yield 75%). The weight average molecular weight M w is 3.0 x 10 4 , polydispersity M w / M n was 1.8.

[0130] [Examples 1 and 2: Preparation of cryoprotectant (A)] The cryoprotectant of the present invention was prepared by adding a polymer to an existing cryoprotectant.

[0131] EuroCollins buffer (EC) contains 17.48 w / v% glucose and potassium dihydrogen phosphate (KH 2 PO 4 ) at 1.02 w / v%, dipotassium hydrogen phosphate (K 2 HPO 4) 3.65 w / v%, potassium chloride (KCl) 0.56 w / v%, sodium bicarbonate (NaHCO 3 EuroCollins buffer solution was prepared by dissolving 0.42 w / v% of 0.42% ethanol in ultrapure water.

[0132] [Table 1]

[0133] EuroCollins buffer solution was added to ultrapure water to a concentration of 20 v / v%, HEPES to a concentration of 0.239 w / v%, and DMSO, FD, and PG were added to the solution to obtain the compositions shown in Table 2, to prepare cryoprotectants VS55, VS50, VS45, and VS40. The pH of each cryoprotectant was adjusted to 8 by adding hydrochloric acid or an aqueous solution of sodium hydroxide.

[0134] [Table 2]

[0135] As shown in Table 3, cryoprotective solutions containing 100 mass%, 90 mass%, or 80 mass% of VS55, VS50, VS45, and VS40, respectively, and 0 mass%, 10 mass%, or 20 mass% of PMB of Production Example 1 were prepared (Reference Example 1, Examples 1-1 to 1-4, Examples 2-1 to 2-4, and Comparative Examples 1-1 to 1-3).

[0136] [Table 3]

[0137] (Cell cryoprotection effect) The cryoprotective effect of the above cryoprotectants on cells was evaluated by cell viability after one freeze-thaw cycle. Mouse pancreatic β cells (MIN6) 4 × 10 5The cells were suspended in 0.2 mL of cryoprotectant in a microtube and quickly immersed in liquid nitrogen to freeze. After freezing, the microtube was removed from the liquid nitrogen and thawed by immersing in a water bath at 37°C. After thawing, 0.2 mL of medium was added and pipetted. 50 μL of the cell suspension was mixed with 50 μL of 0.4% trypan blue staining solution and counted using a Countess. TM Cell viability was measured using an II FL Automated Cell Counter, and the results are shown in Figure 1.

[0138] VS55 showed a good cryoprotective effect on cells, which was consistent with previous reports (Figure 1, Reference Example 1). However, in VS50, VS45, and VS40, the cell viability significantly decreased with decreasing concentrations of DMSO, FD, and PG (Figure 1, Comparative Examples 1-1 to 1-3).

[0139] In contrast, in Examples 1-1 and 2-1, the cell viability could be improved by replacing a part of VS55 with PMB. Furthermore, when PMB was added to VS50, VS45, and VS40, the cell viability could be maintained at the same level as when VS55 was used, despite the reduced concentrations of DMSO, FD, and PG (Examples 1-2 to 1-4). Furthermore, the cryoprotectant containing 20% ​​by mass of PMB exhibited a significantly high cryoprotective effect (Examples 2-1 to 2-4). In other words, it can be said that the cryoprotectant of the present invention can exhibit a significantly high cryoprotective effect compared to the conventional technology, while reducing the amount of DMSO used.

[0140] [Examples 3 to 5: Preparation of cryoprotectant (B)] While maintaining the composition of the existing cryoprotective solution, the cryoprotective solution of the present invention was prepared with a different PMB content. Specifically, EuroCollins buffer solution was added to ultrapure water to give a concentration of 20 v / v% and HEPES to give a concentration of 0.239 w / v%, and DMSO, FD, and PG were added to the solution to give the composition shown in Table 4. The pH was adjusted to 8, and then PMB was added to prepare the cryoprotective solution.

[0141] [Table 4]

[0142] (Cell cryoprotection effect) The cryoprotective effect of cells was evaluated in the same manner as in Examples 1 and 2. A non-frozen group (NF) was prepared as a positive control, and a EuroCollins buffer-added group (EC) was prepared as a negative control. Mouse pancreatic β cells (MIN6) and primary rat hepatocytes were used.

[0143] The results when MIN6 cells were used are shown in Figure 2. VS55 showed a high cryoprotective effect, although the cell viability was lower than that of the unfrozen group. All of the cryoprotective solutions of the present invention containing PMB maintained cell viability at or above that of VS55, and showed a high cryoprotective effect (Examples 3-1 to 5-4). In particular, the cryoprotective solution containing 20% ​​by mass of PMB showed a significantly high cryoprotective effect, and the cell viability was comparable to that of the unfrozen group (Examples 5-1 to 5-5).

[0144] The results when primary rat hepatocytes were used are shown in FIG. 3. From the results of the group not containing PMB, it can be easily understood that hepatocytes are delicate cells. The cryoprotective solution of the present invention containing PMB was able to maintain a high cell viability even when such delicate cells were cryopreserved. In particular, the cryoprotective solution containing 15% by mass or more of PMB showed the same or higher cryoprotective effect as VS55 (Examples 4-1 to 5-5).

[0145] (Magnetite Dispersibility Test) A magnetite dispersion with a magnetite concentration of 5 mg / mL was prepared by mixing magnetite nanoparticles (manufactured by Daiichi High Frequency Industrial Co., Ltd.) with VS55 (Reference Example 1), VS35 (Comparative Example 1-4), or VS35 + 20 wt% PMB (Example 5-4). The prepared dispersion was ultrasonically treated for 60 minutes to disperse the magnetite, and the state of precipitation of magnetite after leaving it for 0, 30, and 60 minutes was photographed.

[0146] The results are shown in Figure 4. The cryoprotective solution of the present invention containing PMB was able to maintain the dispersed state of magnetite even after 30 and 60 minutes (Example 5-4). On the other hand, when PMB was not included, magnetite immediately precipitated at the bottom of the vial within less than 30 minutes. Therefore, it is clear that the addition of PMB allowed magnetite to be dispersed well.

[0147] (Vitrification test) A microtube containing 0.2 mL of the cryoprotective solution of the present invention (Examples 3-1 to 5-4) was immersed in liquid nitrogen and frozen. After freezing, the microtube was taken out of the liquid nitrogen and the state immediately after was photographed.

[0148] The results are shown in Figure 5. EuroCollins buffer solution was not vitrified and was white in color. On the other hand, VS55 was vitrified when frozen, and the transparency of the solution was maintained without whitening (Figure 5). The cryoprotective solution of the present invention showed a tendency to be easily vitrified when the content of the cryoprotectant was high. Specifically, when the content of PMB is A (w / w%) and the total content of dimethyl sulfoxide, formamide and propylene glycol is B (w / v%) relative to the total amount of the cryoprotective solution, it was confirmed that vitrification occurs when 55≦A+B.

[0149] [Manufacturing example 2: PMPC] A PMPC was obtained in the same manner as in Production Example 1, except that a polymerization solution of MPC (1.0 mol / L) and AIBN (0.010 mol / L) in ethanol was used as the polymerization solution for PolyMPC, and the polymerization time was set to 6 hours. w is 1.6 x 10 5 , polydispersity M w / M n was 2.6.

[0150] Comparative Example 2: Study of hydrophobic structural units The effect of the presence of hydrophobic structural units on the cryoprotective effect was examined. Specifically, the cryoprotective effect of the polymer of Production Example 2 was evaluated in the same manner as in Examples 1 to 5. The contents of each cryoprotectant in the cryoprotective solution were the same as in Examples 5-4. Mouse pancreatic β cells (MIN6) and primary rat hepatic parenchymal cells were used.

[0151] The results are shown in Figure 6. For all cells used, PMB from Production Example 1 showed a high cryoprotective effect, whereas the cell survival rate after freezing and thawing was significantly reduced in the cryoprotectant from PMPC (Production Example 2) which does not have a hydrophobic structural unit. From this, it can be seen that the cryoprotectant of the present invention can exhibit a high cryoprotective effect by using a copolymer having both hydrophilic and hydrophobic structural units.

[0152] [Production example 3: PMPC (low molecular weight)] A low molecular weight PMPC was obtained in the same manner as in Production Example 2, except that the concentration of MPC was (0.50 mol / L) and AIBN was (0.020 mol / L). The weight average molecular weight M w is 5.0 x 10 4 , polydispersity M w / M n was 2.6.

[0153] [Manufacturing example 4: PSB] To prepare the polymerization solution of poly(SBMA-co-BMA) (PSB), SBMA (0.15 mol / L), BMA (0.35 mol / L), and AIBN (0.020 mol / L) were added to trifluoroethanol. The polymerization solution was bubbled with argon gas for 15 minutes, sealed, and then polymerized in an oil bath at 60°C for 24 hours. The polymerization solution was reprecipitated in a mixture of diethyl ether and chloroform (9:1 v / v%). The white precipitate was collected by decantation and dried in vacuum to obtain white solid PSB.

[0154] [Manufacturing example 5: PCB] Poly(CBMA-co-BMA) (PCB) was obtained in the same manner as in Production Example 4, except that CBMA was used instead of SBMA as the polymerization solution for poly(CBMA-co-BMA) (PCB).

[0155] [Manufacturing example 6: PPB] As a polymerization solution for Poly(PEGMA-co-BMA) (PPB), PEGMA (number average molecular weight M n = 360), ethanol was used as the polymerization solvent, and the polymerization time was 6 hours, and a PPB was obtained in the same manner as in Production Example 4.

[0156] [ka]

[0157] [Example 6: Study of hydrophilic building blocks] The influence of the type of hydrophilic structural unit on the cryoprotective effect was examined. Specifically, the (co)polymers of Production Examples 1 to 6, which have different hydrophilic structural units, were evaluated for solubility and cryoprotective effect.

[0158] (Solubility test) The (co)polymers of Production Examples 1 to 6 were added to water and VS55 at a ratio of 2 mg / mL, respectively, and their solubility was evaluated. Specifically, the polymers that were completely dissolved were evaluated as ○, and the polymers that were partially or completely undissolved were evaluated as ×.

[0159] The results are shown in Table 5. Of the hydrophilic building blocks that were used other than MPC, only PCB (Production Example 5) was soluble in VS55. On the other hand, PCB was not soluble in water, and PSB (Production Example 4) and PPB (Production Example 6) were not soluble in water or VS55. For this reason, PCB (Production Example 5) was used as a control in the subsequent cryoprotection effect test.

[0160] [Table 5]

[0161] (Cell cryoprotection effect) The cryoprotective effects of the (co)polymers of Production Examples 1 to 3 and 5 were evaluated in the same manner as in Examples 1 to 5. The contents of the cryoprotectants in the cryoprotective solutions used were the same as in Example 5-4. Primary rat hepatocytes were used as the cells.

[0162] The results are shown in Figure 7. The decrease in cell viability with PMPC (Production Examples 2 and 3) is consistent with the experimental results described above. Furthermore, when PCB (Production Example 5) was used, the cell viability after freezing and thawing was significantly decreased compared to the cryoprotective solution of the present invention. From this, and in consideration of the results of the solubility test described above, it can be understood that the cryoprotective solution of the present invention can exhibit a high cryoprotective effect by selecting a specific hydrophilic constituent unit and combining it with a hydrophobic constituent unit.

[0163] [Production Example 7: PMB37 (living radical polymerization)] PMB with different composition ratios was prepared by living radical polymerization. Specifically, ascorbic acid was used as a reducing agent, EBiB was used as an initiator, and CuBr was used as a catalyst. 2 100-mer PMB was synthesized by ARGET-ATRP using TPMA as a ligand. PMB37 was prepared by setting the monomer charge ratio [MPC]:[BMA] to 3:7. Each reagent was added to methanol so that [monomer (MPC+BMA)]=1.0 mol / L, [monomer (MPC+BMA)] / [EBiB] / [ascorbic acid] / [TPMA] / [CuBr2]=100 / 1 / 2 / 0.2 / 0.02. The obtained solution was degassed by bubbling argon gas, and then polymerized for 48 hours in a thermostatic bath at 30°C. The obtained polymerization solution was reprecipitated in a mixed solution of diethyl ether:chloroform=9:1 (v / v%). The white precipitate was collected by decantation and dissolved in pure water. The solution was filtered through a filter with a pore size of 0.45 μm and dialyzed for 3 days with a dialysis membrane of 3.5 kD. The solution was freeze-dried to obtain PMB37 as a white solid. After the reaction, deuterated methanol was used as the solvent. 1H-NMR measurement was performed. As a result, the monomer conversion rate was 90% or more. w is 7.5 x 10 3 , polydispersity M w / M n was 1.4.

[0164] [Production Example 8: PMB55 (living radical polymerization)] PMB55 was obtained in the same manner as in Production Example 7, except that the monomer charging ratio [MPC]:[BMA] was set to 5:5 and the polymerization time was set to 24 hours. The monomer conversion rate was 95% or more. The weight average molecular weight M w is 1.2 × 10 4 , polydispersity M w / M n was 1.5.

[0165] [Production Example 9: PMB73 (living radical polymerization)] PMB73 was obtained in the same manner as in Production Example 7, except that the monomer charging ratio [MPC]:[BMA] was set to 7:3 and the polymerization time was set to 24 hours. The monomer conversion rate was 95% or more. The weight average molecular weight M w is 8.8 x 10 3 , polydispersity M w / M n was 1.5.

[0166] [Production Example 10: PMPC (living radical polymerization)] PMPC was obtained in the same manner as in Production Example 7, except that the monomer charging ratio [MPC]:[BMA] was set to 10:0 and the polymerization time was set to 24 hours. The monomer conversion rate was 95% or more. The weight average molecular weight M w is 1.2 × 10 4 , polydispersity M w / M n was 1.6.

[0167] [Example 7: Examination of composition ratio of structural units] The influence of the composition in the copolymer (hydrophilic constituent units: hydrophobic constituent units) on the cryoprotective effect was examined. Specifically, the (co)polymers of Production Examples 7 to 10, which have different composition ratios, were evaluated for their cryoprotective effect on cells. The contents of each cryoprotectant in the cryoprotective solution were the same as those in Example 5-4. Primary rat hepatocytes were used as the cells.

[0168] The results are shown in Figure 8. The decrease in cell viability in PMPC (Production Example 10) is consistent with the experimental results described above. On the other hand, the change in composition in the copolymer did not have a significant effect on the freezing effect of the cells.

Claims

1. A cryoprotectant for biological samples, comprising: General formula (1): 【Chemistry 1】 [In the formula, R １ R represents a hydrogen atom, a methyl group, or an ethyl group. ２ , R ３ and R ４ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms; L1 represents a single bond, an ether bond, an ester bond, or an amide bond; p and q are the same or different and each represents an integer of 1 to 6. A hydrophilic structural unit represented by the formula: General formula (2): 【Chemistry 2】 [In the formula, R ５ R represents a hydrogen atom, a methyl group, or an ethyl group. ６ represents a hydrocarbon group having one or more carbon atoms; L2 represents a single bond, an ether bond, an ester bond, or an amide bond. A hydrophobic structural unit represented by the formula: The copolymer includes a copolymer having the formula: Cryoprotectant.

2. The total amount of the structural units constituting the copolymer is taken as 100 mol %, The content of the hydrophilic structural unit is 20 to 99 mol %, and The content of the hydrophobic structural unit is 1 to 80 mol %. The cryoprotectant solution of claim 1.

3. The R １ is a hydrogen atom or a methyl group, and The R ５ is a hydrogen atom or a methyl group; The cryoprotectant solution according to claim 1 or 2.

4. The R ２ , R ３ and R ４ The cryoprotectant solution according to claim 1 or 2, wherein is a methyl group.

5. The cryoprotectant according to claim 1 or 2, wherein p and q are the same or different and are integers of 1 to 3.

6. The R ６ The cryoprotectant solution according to claim 1 or 2, wherein is a hydrocarbon group having two or more carbon atoms.

7. The cryoprotectant solution according to claim 1 or 2, wherein L1 and L2 are ester bonds.

8. The cryoprotective solution according to claim 1 or 2, wherein the content of the copolymer is 10% by mass or more, with the total amount of the cryoprotective solution being 100% by mass.

9. Further comprising dimethyl sulfoxide, formamide and propylene glycol; The content of the copolymer relative to the total amount of the cryoprotectant solution is A (w / w%); When the total content of the dimethyl sulfoxide, the formamide and the propylene glycol with respect to the total amount of the cryoprotective solution is B (w / v %), 55≦A+B; The cryoprotectant solution according to claim 1 or 2.

10. The cryoprotectant solution according to claim 1 or 2, wherein the biological sample is a cell, a tissue or an organ.

11. The cryoprotectant solution according to claim 10, wherein the biological sample is hepatic parenchymal cells, liver tissue or liver.

12. 3. The cryoprotectant solution of claim 1 or 2, comprising magnetic nanoparticles.

13. A method for cryopreserving a biological sample, comprising the step of freezing the cryoprotective solution according to claim 1 or 2 and the biological sample.

Images