Cell cryopreservation solution and its use

Abstract

The present invention provides a cell cryopreservation solution that can achieve both a good survival rate and safety for living organisms. The cell cryopreservation solution of the present invention contains propylene glycol and at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose, and is free of dimethyl sulfoxide, thickeners, and natural animal-derived components.

Description

[Technical field]

[0001] The present invention relates to a solution for cell cryopreservation and its use. [Background technology]

[0002] Conventionally, cryopreservation has been performed to prevent cell deterioration due to subculture of cultured cells or contamination by various bacteria, and to utilize cells for a long period of time. A commonly known method for cryopreserving cells is to suspend cells in a solution containing dimethyl sulfoxide (DMSO) or serum, place them in a cryotube or an ampoule, and freeze-preserve them (e.g., in liquid nitrogen). For example, Patent Document 1 describes that cells were cryopreserved using an aqueous cell preservation solution containing DMSO, a thickener, and glucose, and a good survival rate was achieved.

[0003] With the recent development of cell transplantation therapy, the number of opportunities to administer cell preparations to patients is increasing. In the field of cell transplantation (operating room), it is difficult to remove the cryopreservation solution after thawing the cryopreserved cell preparation and before administering it to the patient. Therefore, there is an increasing need for a cell cryopreservation solution that can be safely administered to patients and can provide a sufficient cell survival rate. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent No. 5835853 [Non-patent literature]

[0005] [Non-Patent Document 1] Shu Z, Heimfeld S, Gao D.: Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant. 2014 Apr;49(4):469-76 [Non-Patent Document 2] Alessandrino P, Bernasconi P, Caldera D, Colombo A, Bonfichi M, Malcovati L, Klersy C, Martinelli G, Maiocchi M, Pagnucco G, Varettoni M, Perotti C, Bernasconi C.: Adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: analysis of 126 cases. Bone Marrow Transplant. 1999 Mar;23(6):533-7 Summary of the Invention [Problem to be solved by the invention]

[0006] DMSO is considered to be the best cryoprotectant in terms of cell viability. However, it can affect the expression, differentiation and proliferation of various cell functions. In addition, side effects such as nausea, vomiting, headache, elevated blood pressure, diarrhea and abdominal cramps have been reported in hematopoietic stem cell transplantation using a cell cryopreservation solution containing DMSO (Non-Patent Document 1, Non-Patent Document 2).

[0007] There have been attempts to reduce the amount of DMSO used by combining it with other cryoprotectants (Non-Patent Document 1), but it is still insufficient to ensure both the performance and safety to living organisms by replacing it with another cryoprotectant.

[0008] The present invention has been made to solve such problems, and one of its objects is to provide a solution for cryopreservation of cells that can achieve both a good survival rate and safety for the living body. [Means for solving the problem]

[0009] A cell cryopreservation solution according to one embodiment of the present invention contains at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose, and propylene glycol, and is free of dimethyl sulfoxide, thickeners, and natural animal-derived components.

[0010] A method for cryopreserving cells according to one embodiment of the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells in a state mixed with the above-mentioned cell cryopreservation solution.

[0011] A method for producing frozen cells according to one embodiment of the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells in a state mixed with the above-mentioned cell cryopreservation solution.

[0012] A frozen cell according to one embodiment of the present invention is produced using the method for producing frozen cells described above. Effect of the Invention

[0013] By using a cell cryopreservation solution according to one embodiment of the present invention, the survival rate of cells after thawing is good, and the thawed cells can be safely applied to a living body. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Cell cryopreservation solution] The cell cryopreservation solution of the present invention contains at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose and maltose, and propylene glycol, and does not contain dimethyl sulfoxide (DMSO), thickening agents, or natural animal-derived components.

[0015] The sugar is at least one selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose and maltose. That is, the sugar is one, two, three, four, five or six selected from the group. Glucose is a monosaccharide, mannitol and sorbitol are sugar alcohols, and trehalose, sucrose and maltose are disaccharides. The sugar may be D-isomer, L-isomer or a mixture of D-isomer and L-isomer, but D-isomer is preferred from the viewpoint of production cost and safety. In one example, the sugar is a monosaccharide. In another example, the sugar is a disaccharide. In another example, the sugar is a sugar alcohol. In another example, the sugar is a mixture of any of monosaccharides, disaccharides and sugar alcohols.

[0016] The concentration of the sugar in the cell cryopreservation solution is not particularly limited, but in one example, it is preferably 0.5 w / v% or more, more preferably 1.0 w / v% or more, even more preferably 1.5 w / v% or more, and particularly preferably 2.0 w / v% or more, and is preferably 10.0 w / v% or less, more preferably 6.0 w / v% or less, even more preferably 5.0 w / v% or less, even more preferably 4.5 w / v% or less, and particularly preferably 4.0 w / v% or less. In this specification, the concentration based on "monosaccharide conversion" refers to the concentration calculated by weight for monosaccharides and sugar alcohols, and the concentration calculated assuming that the weight is half for disaccharides. In other words, the actual concentration (w / v%) of the above disaccharides is twice the concentration described above.

[0017] The concentration of propylene glycol in the cell cryopreservation solution is not particularly limited, but in one example, it is preferably 2.5 w / v% or more, more preferably 5.0 w / v% or more, even more preferably 7.5 w / v or more, and particularly preferably 10.0 w / v or more, and from the standpoint of safety for the living body, it is preferably 18.0 w / v% or less, more preferably 16.0 w / v% or less, even more preferably 15.0 w / v% or less, and particularly preferably 13.0 w / v% or less.

[0018] Dimethyl sulfoxide (DMSO) is considered to be the most excellent cryoprotectant in terms of cell viability. However, the above-mentioned problems have been reported. Since the present invention does not contain DMSO, the problems caused by DMSO do not occur. Therefore, the cell cryopreservation solution of the present invention is very useful from the viewpoint of safe application to living bodies, particularly in clinical use. Moreover, as shown in the examples below, by containing propylene glycol, cell viability equivalent to that of DMSO can be achieved.

[0019] Examples of thickeners include carboxymethylcellulose (hereinafter referred to as CMC), sodium carboxymethylcellulose (hereinafter referred to as CMC-Na), organic acid polymers, propylene glycol alginate, and sodium alginate. The cell cryopreservation solution of the present invention does not contain a thickener, so that the occurrence of side effects that may be caused by a thickener can be avoided. Therefore, the cell cryopreservation solution of the present invention is very useful from the viewpoint of being safely applied to living bodies, especially in clinical use. Moreover, as shown in the examples below, cells can be cryopreserved with a good survival rate even without the inclusion of a thickener.

[0020] Examples of natural animal-derived components include albumin, serum, plasma, and basal media. Examples of serum include adult bovine serum, calf serum, newborn calf serum, and fetal bovine serum. Examples of basal media include RPMI medium, MEM medium, HamF-12 medium, and DM-160 medium. Since the cell cryopreservation solution of the present invention does not contain natural animal-derived components, there is no problem of quality differences between lots of natural animal-derived components, and it is possible to avoid the risk of changes in cell properties due to components that are unnecessary for cell preservation, such as various cytokines, growth factors, and hormones contained in serum, and further to avoid the influence of components of unknown origin contained in the basal medium. Therefore, the cell cryopreservation solution of the present invention is very useful from the viewpoint of being safely applied to living bodies, especially in clinical use. Moreover, as shown in the examples described below, cells can be cryopreserved with a good survival rate even without containing natural animal-derived components.

[0021] The cell cryopreservation solution may further contain other components. Examples of the other components include pH adjusters. Examples of the pH adjusters include phosphate buffer and carbonate buffer. In addition, when no phosphate buffer is added to the Basic Stock Solution (BSS), a solution to which physiological saline has been added can also be used. Among these, it is particularly preferable to use a phosphate buffer. It is preferable to use a pH adjuster appropriately to adjust the pH of the cell cryopreservation solution to about 6.5 to 9.0, preferably 7.0 to 8.5. The phosphate buffer in the present invention refers to sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate (anhydrous), potassium chloride, and potassium dihydrogen phosphate (anhydrous), and it is particularly preferable to use sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous). In addition, a combination of potassium dihydrogen phosphate, sodium chloride, potassium chloride, and disodium hydrogen phosphate is also preferable. The pH adjuster is preferably contained in the cell cryopreservation solution at 0.01 to 1.0 w / v %, more preferably at 0.05 to 0.5 w / v %. The composition of the inorganic salt solution used in the examples is also suitable.

[0022] The cell cryopreservation solution is preferably an aqueous solution. In order to maintain the performance as a preservation solution, the osmotic pressure of the cell cryopreservation solution is preferably 1000 mOsm or more, and more preferably 1000 to 2700 mOsm.

[0023] The composition of the cell cryopreservation solution may be any combination of the specific components listed above, as long as the composition is capable of adequately preserving cells. The concentrations may also be selected and combined. Furthermore, the components and / or concentrations in the examples described below may also be selected and combined. That is, the individual components and / or concentrations disclosed in this specification may be selected and combined.

[0024] In a preferred embodiment, the cell cryopreservation solution is an aqueous solution that contains sugars and propylene glycol, but does not contain DMSO, thickeners, or natural animal-derived components. In a more preferred embodiment, the cell cryopreservation solution is an aqueous solution that contains sugars, propylene glycol, and a pH adjuster, but does not contain DMSO, thickeners, or natural animal-derived components. In a further preferred embodiment, the cell cryopreservation solution is an aqueous solution that contains only sugars, propylene glycol, and a pH adjuster, but does not contain DMSO, thickeners, or natural animal-derived components (i.e., it is composed of only sugars, propylene glycol, a pH adjuster, and water).

[0025] It is preferable that the cell cryopreservation solution is sterilized, since the risk of infection by bacteria, etc. is reduced, and therefore the solution can be applied to a living body more safely. In addition, in research applications, the risk of contamination by bacteria, etc. is reduced.

[0026] Glucose, mannitol, sorbitol, trehalose, sucrose and maltose have been used as components of intravenous injections. In particular, 5% glucose injections are usually administered intravenously at 500-1000mL per dose for adults for water supply, drug or poison poisoning, and liver disease. As such, the above six sugars are extremely safe for the living body.

[0027] Propylene glycol is also used as a solubilizer in injections when the active ingredient is poorly soluble in the solvent. According to safety data from the Japan Pharmaceutical Excipients Association, the maximum dosage for the human body is 3.2g for intravenous injection. Thus, propylene glycol is extremely safe for the living body.

[0028] The cells to be cryopreserved are not particularly limited, and examples thereof include cell lines, lymphocytic cells, spleen cells, thymus cells, fertilized eggs, hematopoietic stem cells, adult stem cells, mesenchymal stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells) of various organisms. Organisms from which the cells are derived include humans and non-human animals, and more specifically, insects, fish, amphibians, reptiles, birds, and mammals. Mammals include laboratory animals such as mice, rats, rabbits, guinea pigs, and primates other than humans; pet animals such as dogs and cats; livestock such as pigs, cows, goats, sheep, and horses; and humans.

[0029] The cell cryopreservation solution of the present invention can achieve a good viability, for example, after one week or more (for example, after 10 years or more) of storage, depending on the cells to be preserved. In one example, the viability after thawing can be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0030] The cell cryopreservation solution of the present invention has the advantage of being capable of achieving a good survival rate, and therefore can be used as an excellent cell cryopreservation solution even in cases other than those in vivo (e.g., for research purposes).

[0031] The cell cryopreservation solution of the present invention may be combined with, for example, a cold-resistant container, a temperature-controllable freezer or slow-freezing container, or an instruction manual, etc., to be made into a kit. The instruction manual of the kit records, for example, at least a part of the contents of the cryopreservation method of the present invention described in the section [Method for cryopreserving cells] below and / or the contents of the production method of the present invention described in the section [Method for producing frozen cells] below.

[0032] [Method of cryopreserving cells] The method for cryopreserving cells according to the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells in a state mixed with the cell cryopreservation solution.

[0033] In the mixing step, the number of cells per 1 mL of the cell cryopreservation solution is not particularly limited, but is preferably 10 3 ~10 9 / mL, more preferably 10 4 ~10 8 It is preferable to prepare the solution so that the concentration becomes 1 / mL.

[0034] Before the mixing step, the cells may be washed with a washing solution such as PBS (phosphate buffered saline), which can further reduce contamination with components of the culture medium, for example.

[0035] The mixture may be transferred to a cold-resistant container, such as, for example, an ampoule or a cryotube, which is preferably internally sterile.

[0036] In the freezing step, the cooling rate is not particularly limited, but from the viewpoint of increasing the survival rate, it is preferable to cool slowly, and more preferably at a cooling rate of about -1°C / min. Therefore, the freezing step is preferably performed using a temperature-controllable freezer or slow freezing container. Examples of slow freezing containers include CoolCell (registered trademark) from Corning, Mr. Frosty from Nalgene, and Bycell from Nippon Freezer Co., Ltd. The final freezing temperature is not particularly limited, but is preferably -80°C or lower, more preferably -150°C or lower, and even more preferably -196.5°C or lower. After storing at around -80°C, it may be transferred to -180°C to -200°C (for example, in liquid nitrogen) for storage.

[0037] As described above, cells cryopreserved using the cryopreservation method of the present invention can exhibit a good survival rate after thawing (see also the Examples described below). Thawing is preferably performed quickly, for example, by immersing the cells in a water bath at 37°C ± 1°C. When using the cells after thawing, the cell cryopreservation solution may or may not be removed.

[0038] In one example, the thawed mixture can be applied to a living body without removing the cell cryopreservation solution. The cell cryopreservation solution of the present invention can be composed of components that are very safe for living bodies, so it can be directly applied to a living body. Therefore, it can be possible to perform cell transplantation easily and quickly at the site of cell transplantation (operating room). In another example, the thawed mixture can be added to a culture medium or the like, and the cell cryopreservation solution can be removed by centrifugation, washing, or the like before use. The liquid used for washing may be appropriately selected depending on the subsequent processing content and the use of the cells, and for example, culture medium, physiological saline, PBS, or the like may be used. For example, in research situations where components during cell culture need to be strictly controlled, it may be preferable to remove the cell cryopreservation solution. In another example, the thawed mixture can be used in experiments such as cell culture without removing the cell cryopreservation solution. The cell cryopreservation solution of the present invention can be composed of components that are very safe for cells, so cell culture can be performed suitably by diluting it with a culture medium or the like without washing.

[0039] [Method for preparing frozen cells and frozen cells] The method for producing frozen cells according to the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells while mixed with the cell cryopreservation solution.

[0040] The specific method for preparing frozen cells is the same as that described above in the section "Method for cryopreserving cells."

[0041] As described above, the frozen cells produced by the production method according to the present invention can exhibit a good survival rate after thawing (see also the Examples described below). In one example, the survival rate after thawing can be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0042] The frozen material may be, for example, a cell preparation, or a normal or cancer tissue having a size of a few centimeters or less, etc. Preferable examples of cells contained in the cell preparation include mesenchymal stem cells, hematopoietic stem cells, lymphocytic cells, dendritic cells, neural cells, keratinocytes, and fibroblasts.

[0043] The following examples are provided to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated by reference.

[0044] 〔summary〕 A cell cryopreservation solution according to one embodiment of the present invention contains at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose, and propylene glycol, and is free of dimethyl sulfoxide, thickeners, and natural animal-derived components.

[0045] In the cell cryopreservation solution, the concentration of the propylene glycol is preferably 5.0 w / v % or more.

[0046] In the cell cryopreservation solution, the concentration of the propylene glycol is more preferably 10.0 w / v % or more and 15.0 w / v % or less.

[0047] In the cell cryopreservation solution, the concentration of the sugars is preferably 1.0 w / v % or more and 5.0 w / v % or less in terms of monosaccharides.

[0048] In the above-mentioned cell cryopreservation solution, the concentration of the above-mentioned sugars is preferably 2.0 w / v % or more and 4.0 w / v % or less in terms of monosaccharides.

[0049] The above-mentioned cell cryopreservation solution preferably further contains a pH adjuster.

[0050] A method for cryopreserving cells according to one embodiment of the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells in a state mixed with the above-mentioned cell cryopreservation solution.

[0051] A method for producing frozen cells according to one embodiment of the present invention includes a mixing step of mixing the above-mentioned cell cryopreservation solution with cells, and a freezing step of freezing the cells in a state mixed with the above-mentioned cell cryopreservation solution.

[0052] In the above method, the freezing step is preferably carried out using a temperature-controllable freezer or slow-freezing container.

[0053] A frozen cell according to one embodiment of the present invention is produced using the method for producing frozen cells described above. EXAMPLES

[0054] <Cells used> Jurkat: Human T-cell leukemia cell line P3U1: Mouse myeloma cell line Mesenchymal stem cells (MSC) (obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) iPS cells (induced pluripotent stem cells): 201B7 strain (obtained from iPS Academia Japan, Inc.)

[0055] <Test Method> [Cryopreservation] 1. Various cells were harvested from culture flasks, and viable cells were counted by trypan blue staining. 2. The required amount of cell suspension was dispensed into a 15 mL Falcon tube and centrifuged at 1,200 rpm for 5 minutes (4°C), after which the supernatant was removed. 3. 3 mL of each cell cryopreservation solution was added (iPS cells: 0.6 mL) and the pellet was suspended. 4. The cell suspension was dispensed into 1.5 mL cryotubes at 1 mL (iPS cells: 0.2 mL). The number of cells was 3 x 10 6 cells / tube (Jurkat), 1×10 6 cells / tube (MSC and P3U1), or 2×10 5 cells / tube (iPS). 5. The cryotube was placed in a pre-cooled Mr. Frosty (Nalgene) and frozen overnight in a -80°C freezer. 6. The next day, the cryotubes were transferred to a -152°C freezer and frozen for at least one week.

[0056] [Unzip] 1. Remove the cryotube from the -152°C freezer and quickly thaw it in a 37°C water bath. 2. 9 mL of medium that had been brought to room temperature was placed in a 15 mL Falcon tube, and the thawed cell suspension was transferred thereto. 3. After centrifugation at 1,200 rpm for 5 minutes (4°C), the supernatant was removed. 4. 3 mL (Jurkat), 1 mL (MSC and P3U1), or 0.2 mL (iPS cells) of medium was added. 5. Viable cells were counted by trypan blue staining, and the cell viability was calculated.

[0057] <Preparation of cell cryopreservation solution> [Inorganic salt solution] The following substances were dissolved in distilled water at the following concentrations to prepare an inorganic salt solution. This inorganic salt solution has a pH adjusting function. Potassium dihydrogen phosphate 0.0328 g / L Sodium chloride 1.315 g / L Potassium chloride 0.0328 g / L Disodium hydrogen phosphate dodecahydrate 0.189 g / L

[0058] [Cell cryopreservation solution] In each experiment, each component was added to an inorganic salt solution so as to obtain the composition described below, and mixed. This was then filtered to prepare a sterile solution for cell cryopreservation.

[0059] <Experiment 1> The cryopreservation effects of DMSO, glycerol and propylene glycol in combination with glucose were investigated.

[0060] Solution 1-A: 3.0 w / v% glucose Solution 1-B: 3.0 w / v% glucose + 10.0 w / v% DMSO (MW 78.13) Solution 1-C: 3.0 w / v% glucose + 10.0 w / v% glycerol (MW 92.09) Solution 1-D: 3.0 w / v% glucose + 10.0 w / v% propylene glycol (MW 76.09)

[0061] [Table 1]

[0062] Compared to glycerol, propylene glycol was shown to achieve viability comparable to that of DMSO.

[0063] <Experiment 2> The concentration dependency of the cryopreservation effect of propylene glycol was investigated.

[0064] Solution 2-A: 2.5 w / v% propylene glycol + 3 w / v% glucose Solution 2-B: 5.0 w / v% propylene glycol + 3 w / v% glucose Solution 2-C: 7.5 w / v% propylene glycol + 3 w / v% glucose Solution 2-D: 10.0 w / v% propylene glycol + 3 w / v% glucose Solution 2-E: 12.5 w / v% propylene glycol + 3 w / v% glucose Solution 2-F: 15.0 w / v% propylene glycol + 3 w / v% glucose

[0065] [Table 2]

[0066] <Experiment 3> The cryopreservation effects of various sugars in combination with propylene glycol were investigated.

[0067] Solution 3-A: 10.0 w / v% propylene glycol Solution 3-B: 3.0 w / v% glucose (MW 180.2) + 10.0 w / v% propylene glycol Solution 3-C: 3.0 w / v% mannitol (MW 182.2) + 10.0 w / v% propylene glycol Solution 3-D: 3.0 w / v% sorbitol (MW 182.2) + 10.0 w / v% propylene glycol Solution 3-E: 6.0 w / v% trehalose (MW 342.3) + 10.0 w / v% propylene glycol Solution 3-F: 6.0 w / v% sucrose (MW 342.3) + 10.0 w / v% propylene glycol Solution 3-G: 6.0 w / v% maltose (MW 342.3) + 10.0 w / v% propylene glycol

[0068] [Table 3]

[0069] It was shown that high survival rates could be achieved on any of glucose, mannitol, sorbitol, trehalose, sucrose and maltose.

[0070] <Test 4> The concentration dependence of the cryopreservation effect of sugar (glucose) was investigated.

[0071] Solution 4-A: 10.0 w / v% propylene glycol Solution 4-B: 1.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-C: 2.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-D: 3.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-E: 4.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-F: 5.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-G: 6.0 w / v% glucose + 10.0 w / v% propylene glycol

[0072] [Table 4]

[0073] <Test 5> The cryopreservation effect of various sugars in the absence of propylene glycol was examined.

[0074] Solution 5-A: 10.0 w / v% propylene glycol + 3.0 w / v% glucose (MW 180.2) Solution 5-B: 3.0 w / v% glucose (MW 180.2) Solution 5-C: 3.0 w / v% mannitol (MW 182.2) Solution 5-D: 3.0 w / v% sorbitol (MW 182.2) Solution 5-E: 6.0 w / v% trehalose (MW 342.3) Solution 5-F: 6.0 w / v% sucrose (MW 342.3) Solution 5-G: 6.0 w / v% maltose (MW 342.3)

[0075] [Table 5]

[0076] In the absence of propylene glycol, the survival rate was very low for all sugars. [Industrial Applicability]

[0077] The present invention can be used for cryopreservation of cells in medical treatment, research, and the like.

Claims

1. A solution for cell cryopreservation, comprising glucose, propylene glycol and a pH adjuster, and not containing dimethyl sulfoxide, a thickener and natural animal-derived components; The concentration of the propylene glycol is 10.0 w / v% or more and 15.0 w / v% or less, The glucose concentration is 2.0 w / v% or more and 4.0 w / v% or less, The osmotic pressure of the cell cryopreservation solution is 1490 mOsm or more and 3080 mOsm or less, The pH adjuster is a phosphate buffer solution. Cell cryopreservation solution.

2. A solution for cell freezing preservation as described in claim 1, wherein the phosphate buffer solution consists of a combination of potassium dihydrogen phosphate, sodium chloride, potassium chloride and disodium hydrogen phosphate.

3. 1. A method for cryopreserving cells, comprising: A mixing step of mixing the cell cryopreservation solution according to claim 1 or 2 with cells; and freezing the cells in admixture with the cell cryopreservation solution.

4. 1. A method for producing a frozen product of cells, comprising the steps of: A mixing step of mixing the cell cryopreservation solution according to claim 1 or 2 with cells; and freezing the cells in admixture with the cell cryopreservation solution.

5. The method according to claim 3 or 4, wherein the freezing step is carried out using a temperature-controllable freezer or slow-freezing vessel.

6. A frozen cell product produced using the method of claim 4.

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