Cell freezing composition, cell freezing method, cell culture method, and cell freezing kit

Abstract

The composition for freezing cells according to the present invention comprises a culture medium, an anti-freezing agent and dissolved hydrogen.

Description

[Technical Field]

[0001] The present invention relates to a composition for freezing cells, a method for freezing cells, a method for culturing cells, and a kit for freezing cells. [Background technology]

[0002] Various cell freezing techniques have been developed to date. One such technique is known from Patent Document 1. Patent Document 1 describes a cryopreservation medium composed of 90% fetal bovine serum and 10% DMSO (see, for example, paragraph 0073 of Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2012-512637 Summary of the Invention [Problem to be solved by the invention]

[0004] However, as a result of investigations by the present inventors, it was found that the cryopreservation medium described in Patent Document 1 above has room for improvement in terms of cell proliferation ability after freeze-thawing. [Means for solving the problem]

[0005] A common cell freezing method uses a cell freezing medium containing a cryoprotectant such as DMSO in the cell culture medium. Cells are placed in the cell freezing medium and frozen. The frozen cells are then thawed, cultured, and mass-produced. In this conventional method, if cells are damaged by low-temperature stress during freezing and thawing, their survival activity and proliferation rate may decrease, resulting in a decrease in cell proliferation ability.

[0006] Taking the above circumstances into consideration, the inventors conducted extensive research into the environment within cell freezing preservation solutions, and discovered that by adding hydrogen water and / or hydrogen gas to cell freezing preservation solutions to contain dissolved hydrogen, the decline in cell proliferation ability of cells after freezing and thawing can be suppressed, leading to the completion of the present invention.

[0007] According to the present invention, A culture medium; antifreeze agent, Dissolved hydrogen, A composition for freezing cells is provided, comprising:

[0008] Further, according to the present invention, A culture medium; antifreeze agent, A composition for freezing cells, comprising: The redox potential of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is -700 mV or more and -100 mV or less; The pH of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is 6.8 or more and 8.5 or less. A composition for freezing cells is provided.

[0009] Further, according to the present invention, preparing a mixture of the cell freezing composition and cells; freezing the mixture to obtain a frozen product; A method for freezing cells is provided, comprising:

[0010] Further, according to the present invention, A step of thawing the frozen product obtained by the above-mentioned cell freezing method to obtain a thawed product; Culturing the cells contained in the resulting lysate; A cell culture method is provided, comprising:

[0011] The present invention also provides a cell freezing kit comprising a storage container containing hydrogen water. [Effects of the Invention]

[0012] According to the present invention, there are provided a cell freezing composition that exhibits excellent cell proliferation ability after freezing and thawing, a cell freezing method using the same, a cell culture method, and a cell freezing kit. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a graph showing MTT activity values ​​per cell in Example 1 and Comparative Example 1. [Figure 2] FIG. 1 shows cell growth curves in Example 1 and Comparative Example 1. [Figure 3] FIG. 1 shows MTT activity values ​​in Examples 3 to 5 and Comparative Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0014] An outline of the cell freezing composition of this embodiment will be described.

[0015] One of the compositions for cell freezing of this embodiment contains a culture medium, a cryoprotectant, and dissolved hydrogen.

[0016] According to the findings of the present inventors, it has been discovered that by adding hydrogen water and / or hydrogen gas to a cell freezing composition containing a cell culture medium containing cell nutrients and a cryoprotectant to contain dissolved hydrogen, it is possible to increase the cell viability and cell proliferation rate of the cells after freezing and thawing, thereby suppressing a decrease in cell proliferation ability.

[0017] Dissolved hydrogen refers to hydrogen molecules dissolved in a liquid. When a dissolved hydrogen meter is used, a dissolved hydrogen concentration of, for example, 0.1 ppm or more is measured at atmospheric pressure and 25°C, and the liquid is considered to contain dissolved hydrogen.

[0018] Although the detailed mechanism is unclear, it is thought that a relatively high concentration of dissolved hydrogen in the cell freezing composition can promote cell activity during mixing before the freezing operation, thereby reducing damage to cells during the freezing process. Another possible factor in reducing cell damage is the effect of dissolved hydrogen's ability to remove active oxygen.

[0019] By using the composition for cell freezing of this embodiment, a cell freezing method that can obtain cells with excellent cell proliferation ability, i.e., MTT activity, after freezing and thawing, and a cell culture method using such cells can be realized.Furthermore, a cell freezing method and cell culture method that can obtain cells with excellent cell proliferation rate can be realized. This method can be used for storing and transporting organs and is beneficial for medical technologies such as regenerative medicine.

[0020] Each component of the composition for cell freezing of this embodiment will be described in detail below.

[0021] The cell freezing composition is a hydrogen-containing cell freezing preservative solution used to freeze cells. The cell includes at least one of a single cell, a plurality of cells, a tissue made up of a collection of cells, and an organ.

[0022] Cells used for cell freezing can be, for example, animal cells or plant cells.

[0023] Examples of animal cells include invertebrate-derived cells such as insect cells, mammalian-derived cells such as human cells and mouse cells, and vertebrate-derived cells such as bird-derived cells, amphibian-derived cells, reptile-derived cells, and fish-derived cells. Among these, mammalian-derived cells may be used. Examples of mammals include primates such as humans, mice, guinea pigs, rats, cows, horses, pigs, goats, dogs, and rabbits.

[0024] Animal cells may be in any form, such as primary cultured cells, established cell lines, passaged cells, cultured cells, somatic cells, stem cells, tumor cells, etc. Stem cells include adult stem cells, ES cells, and iPS cells.

[0025] More specific examples of cells include: ES cells, iPS cells, Mesenchymal cells, fibroblasts, muscle cells, bone cells, adipocytes, nerve cells, epithelial cells, Mesenchymal stem cells, fibroblast stem cells, muscle stem cells, bone stem cells, adipose stem cells, neural stem cells, epithelial stem cells, Cells induced to differentiate from ES cells, cells induced to differentiate from iPS cells, cells induced to differentiate from mesenchymal stem cells, cells induced to differentiate from fibroblast stem cells, cells induced to differentiate from muscle stem cells, cells induced to differentiate from bone stem cells, cells induced to differentiate from adipose stem cells, cells induced to differentiate from neural stem cells, or cells induced to differentiate from epithelial stem cells The cells may be adherent cells or suspension cells.

[0026] The composition for cell freezing includes a culture medium, which may contain components necessary for the survival of cells.

[0027] The medium is not particularly limited as long as it contains nutrients for the cells, and examples thereof include freezing medium, complete medium, medium containing serum, serum-free medium, etc. In the case of cells used in regenerative medicine, a serum-free medium may be used.

[0028] Specific examples of the medium include known basal media, such as DMEM medium, EMEM medium, RPMI-1640 medium, α-MEM medium, F-12 medium, F-10 medium, M-199 medium, HAM medium, ERDF medium, L-15 medium, and Williams E medium. These may be used alone or in combination of two or more.

[0029] The cell freezing composition contains a cryoprotectant, which protects cells from cell damage caused by ice crystals and osmotic pressure.

[0030] The antifreeze agent is not particularly limited, and known agents may be used, specifically dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol (EG), glycerol, etc. These may be used alone or in combination of two or more.

[0031] The concentration of the cryoprotectant is appropriately selected depending on the type of cryoprotectant and the cell type, but may be, for example, 1% (v / v) to 15% (v / v), and preferably 5% (v / v) to 10% (v / v), relative to the total medium.

[0032] The composition for cell freezing may be configured to contain hydrogen water. Hydrogen water is water in which hydrogen is dissolved, and refers to water that has a dissolved hydrogen concentration equal to or higher than a specified level at atmospheric pressure and 25°C.

[0033] Hydrogen water can be prepared using raw water by various methods, such as pressurized dissolution, swirling flow, ultrasonic, and micropore methods. Among these, the pressurized dissolution method may be used. By selecting an appropriate method, the dissolved hydrogen concentration and dissolved oxygen concentration can be appropriately controlled. For example, the pressurized dissolution method can increase the dissolved hydrogen concentration in water while reducing the dissolved oxygen concentration.

[0034] The raw water may be distilled water, ion-exchanged water, ultrapure water, etc. These waters may be sterilized by heat treatment, filtering, etc.

[0035] The lower limit of the dissolved hydrogen concentration in hydrogen water when measured under atmospheric pressure at 25°C is, for example, 0.1 ppm or more, preferably 0.2 ppm or more, and more preferably 0.3 ppm or more. In contrast, the dissolved hydrogen concentration in raw water placed in an open environment so as to come into contact with the atmosphere is usually 0.0 ppm under atmospheric pressure at 25°C. On the other hand, the upper limit of the dissolved hydrogen concentration in hydrogen water may be, for example, 10.0 ppm or less, 5.0 ppm or less, preferably 2.0 ppm or less, 1.6 ppm or less, or 1.5 ppm or less.

[0036] The upper limit of the dissolved oxygen concentration of hydrogen water when measured under atmospheric pressure at 25° C. is, for example, 9.0 ppm or less, preferably 5.0 ppm or less, and more preferably 3.0 ppm or less. On the other hand, the lower limit of the dissolved oxygen concentration of hydrogen water is not particularly limited, but may be 0.0 ppm or more.

[0037] The hydrogen water may contain fine bubbles such as ultrafine bubbles of 1 μm or less and microbubbles of more than 1 μm and 100 μm or less, but may also be configured not to contain these fine bubbles.

[0038] In addition to the above components, the composition for cell freezing may contain one or more appropriate serums and additives as needed, as long as the effects of the present invention are not impaired.

[0039] Examples of additives include amino acids, non-essential amino acids, vitamins, low molecular weight compounds, sugars, proteins, buffers, inorganic salts, antibiotics, antibacterial agents, antioxidants, antifreeze agents, etc. These may be used alone or in combination of two or more.

[0040] As the serum, for example, animal serum may be used, and specific examples include human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, and rat serum.

[0041] Examples of buffers include PBS, HEPES, MES, and HANK'S.

[0042] The method for preparing the composition for cell freezing is not particularly limited, but it can be obtained by mixing the above-mentioned components. The order in which the components are added can be changed as appropriate and is not limited. For example, hydrogen water, culture medium, and cryoprotectant may be mixed, or the cryoprotectant may be added to a previously prepared mixture of hydrogen water and culture medium. Alternatively, hydrogen gas may be added to the medium beforehand and then mixed with other components, or hydrogen gas may be added to a mixture of the medium and other components.

[0043] The composition for cell freezing obtained as described above has the following properties.

[0044] The upper limit of the redox potential of the composition for cell freezing, when measured under atmospheric pressure at 25°C, is, for example, -100 mV or less, preferably -150 mV or less, and more preferably -200 mV or less. This increases the cell proliferation ability after freezing and thawing. On the other hand, the lower limit of the redox potential of the composition for cell freezing may be, for example, -700 mV or more, or -600 mV or more.

[0045] The lower limit of the dissolved hydrogen concentration of the composition for cell freezing, when measured under atmospheric pressure at 25°C, is, for example, 0.1 ppm or more, preferably 0.2 ppm or more, and more preferably 0.3 ppm or more. This increases the cell proliferation ability after freezing and thawing. On the other hand, the upper limit of the dissolved hydrogen concentration of the composition for cell freezing may be, for example, 10.0 ppm or less, 5.0 ppm or less, preferably 2.0 ppm or less, 1.6 ppm or less, or 1.5 ppm or less.

[0046] The upper limit of the dissolved oxygen concentration of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is, for example, 9.0 ppm or less, preferably 5.0 ppm or less, and more preferably 3.0 ppm or less. This increases the cell proliferation ability after freezing and thawing. On the other hand, the lower limit of the dissolved oxygen concentration of the composition for cell freezing is not particularly limited, but may be 0.0 ppm or more.

[0047] When measured under atmospheric pressure at 25°C, the lower limit of the pH of the composition for cell freezing is, for example, 6.8 or higher, preferably 7.0 or higher. On the other hand, the upper limit of the pH of the composition for cell freezing is, for example, 8.5 or lower, preferably 8.4 or lower. By keeping the pH within the above numerical range, an environment suitable for cell culture can be achieved.

[0048] Another example of the composition for cell freezing of this embodiment is a composition for cell freezing that contains a culture medium and a cryoprotectant, and that has an oxidation-reduction potential of -700 mV or more and -100 mV or less when measured at atmospheric pressure and 25°C, and a pH of 6.8 or more and 8.5 or less when measured at atmospheric pressure and 25°C. In this case, the oxidation-reduction potential and pH of the composition for cell freezing may be appropriately combined with the above-mentioned upper and lower limits.

[0049] According to this embodiment, by appropriately adjusting the numerical range of the redox potential and the numerical range of the pH of the composition for cell freezing, it is possible to suppress the decrease in cell proliferation ability after freezing and thawing of cells frozen and thawed using the composition for cell freezing.

[0050] In this embodiment, the above redox potential, dissolved hydrogen concentration, dissolved oxygen concentration, and pH can be controlled by, for example, appropriately selecting the type and amount of each component contained in the composition for cell freezing, the method for preparing the composition for cell freezing, etc. Among these, factors for setting the above redox potential, dissolved hydrogen concentration, dissolved oxygen concentration, and pH within the desired numerical range include, for example, using hydrogen water whose dissolved hydrogen concentration is supersaturated, saturated, or close to saturated, appropriately adjusting the storage conditions of the hydrogen water, and the time between preparation of the composition for cell freezing and adding the cells, etc.

[0051] The redox potential, dissolved hydrogen concentration, dissolved oxygen concentration, and pH of the composition for cell freezing are values ​​measured at room temperature (23°C) and atmospheric pressure, and are preferably values ​​measured immediately before use for cell freezing. "Immediately before" means, for example, within 1 hour, preferably within 30 minutes, and more preferably within 15 minutes. In particular, the dissolved hydrogen concentration is preferably the value immediately before use for cell freezing, since the concentration gradually decreases over time when hydrogen water / cell freezing composition is left in an open environment.

[0052] The saturated hydrogen concentration of water at room temperature and pressure is 1.6 ppm, but by adding hydrogen gas to water and / or culture medium under pressurized conditions, it is possible to prepare hydrogen water / cell freezing composition with a supersaturated dissolved hydrogen concentration. Hydrogen gas can be added under pressure in a container such as an aluminum pouch, resin bag, or resin box. By keeping the dissolved hydrogen concentration below the upper limit, hydrogen water / cell freezing composition can be prepared that does not affect the frozen cells or the operator (human). Furthermore, the operator can prepare hydrogen water / cell freezing composition with a predetermined dissolved hydrogen concentration near the site of the cell freezing operation.

[0053] The hydrogen water / cell freezing composition having a predetermined dissolved hydrogen concentration can be prepared by the operator near the work site immediately before cell freezing. Alternatively, hydrogen water produced at another facility, stored in a storage container, and transported may be used to prepare a composition for cell freezing.

[0054] The cell freezing method and cell culture method of this embodiment will be described.

[0055] The devices and instruments used for freezing, storing and cooling the cells, as well as the various reagents and components used therefor, may be sterilized as necessary.

[0056] The cell freezing method includes the steps of preparing a mixture of the above-mentioned cell freezing composition and cells, and freezing the mixture to obtain a frozen product. The mixture can be obtained, for example, by suspending cells in the cell freezing composition.

[0057] The cells may be obtained, for example, by recovering subcultured cells. Adherent cells can be detached from the surface of the cell container and recovered as a precipitate by centrifugation in a specific medium. Suspension cells can be recovered as a precipitate by centrifugation of the suspension in the culture container.

[0058] The density of the cells to be seeded varies depending on the type of cells, but for example, it is about 1 × 10 3 cells / mL ~ approx. 1×10 9 cells / mL, more preferably about 1 x 10 4 cells / mL ~ approx. 1×10 7 May also be expressed as cells / mL.

[0059] Cell freezing may be performed according to a protocol for freezing cells. An example of a cell freezing protocol is shown below. (1) Cells are suspended in culture medium and the suspension is placed in a 15 mL conical tube. (2) Add serum and dimethyl sulfoxide (DMSO) to the cell suspension at a ratio of 7:2:1. (3) Dispense 1 mL of the mixture obtained in (2) above into a cryotube. (4) Cryopreserving the cells. In the above protocol, hydrogen water may be added to the suspension in step (1), or may be added together with serum in step (2). Hydrogen water can be added in step (1) because it makes it easier to adjust the concentrations of each component, serum, and DMSO in the suspension.

[0060] The method for preparing the composition for cell freezing is not particularly limited, but at least one of the following methods can be used: mixing hydrogen water with a culture medium, adding hydrogen water to a cryoprotectant, adding hydrogen water to a mixture containing a culture medium and a cryoprotectant, adding hydrogen gas to a culture medium, adding hydrogen gas to a cryoprotectant, and adding hydrogen gas to a mixture containing a culture medium and a cryoprotectant. In the method of mixing hydrogen water, components such as hydrogen water, culture medium, and antifreeze agent may be mixed, or components such as antifreeze agent may be added to a mixture of hydrogen water and culture medium that has been prepared in advance. In the case of the method of adding hydrogen gas, hydrogen gas may be added to each component such as the culture medium or the antifreeze agent, and then mixed with other components, or hydrogen gas may be added to a mixture containing at least one or both of the culture medium and the antifreeze agent.

[0061] The hydrogen water may be used immediately after preparation, or may be stored in a sealed container filled with hydrogen gas. The hydrogen gas may be added in the atmosphere or under pressure in a closed space.

[0062] Cells may be frozen under gentle conditions at a cooling rate of about -1°C / min.

[0063] The cells may be stored, for example, in an environment at or below −80° C., preferably at or below −130° C., more preferably at or below −150° C. For example, they can be stored in a liquid nitrogen storage container.

[0064] The cell culture method includes a step of thawing the frozen product obtained by the cell freezing method to obtain a thawed product; and culturing the cells contained in the resulting lysate.

[0065] The method for thawing cells varies depending on the cell freezing composition used for freezing and the type of cells, but for example, the frozen material may be exposed to an environment of 0°C to 40°C, preferably about 36°C to 37°C, for example, for 30 seconds to 10 minutes. For example, the frozen material may be immersed in a water bath at about 37°C. This allows the cells in the frozen material to be thawed appropriately.

[0066] The frozen and thawed cells are cultured. Culture conditions vary depending on the cell type being cultured, and can be performed under commonly used conditions. For example, mammalian cells may be cultured at 37°C in a 5% CO2 atmosphere, without particular limitation.

[0067] The cell freezing kit of this embodiment will be described.

[0068] The cell freezing kit contains a cell freezing composition for use in a cell freezing method or components used to prepare a cell freezing composition.

[0069] One of the cell freezing kits includes at least a storage container containing hydrogen water. The storage container may contain hydrogen water in a liquid phase and hydrogen gas in a gas phase. By filling the gas phase with hydrogen gas, the dissolved hydrogen concentration in the hydrogen water can be maintained at a certain level or higher. The storage temperature may be, for example, 0°C to 30°C, preferably 23°C to 27°C.

[0070] The lower limit of the dissolved hydrogen concentration of hydrogen water measured immediately after opening the storage container under atmospheric pressure at 25° C. is, for example, 0.1 ppm or more, preferably 0.2 ppm or more, and more preferably 0.3 ppm or more. On the other hand, the upper limit of the dissolved hydrogen concentration of the hydrogen water may be, for example, 10.0 ppm or less, 5.0 ppm or less, preferably 2.0 ppm or less, 1.6 ppm or less, or 1.5 ppm or less.

[0071] The storage container may be made of a material with low hydrogen permeability or oxygen permeability, for example, an aluminum bag.

[0072] Users can prepare a cell freezing composition using hydrogen water removed from the storage container in the cell freezing kit, which can enhance cell proliferation ability after freezing and thawing.

[0073] The storage container in the cell freezing kit may contain at least a mixture of hydrogen water and a culture medium. Even when hydrogen water is used as a mixed liquid, the cell proliferation ability after freezing and thawing can be enhanced by using a cell freezing composition containing hydrogen water.

[0074] The cell freezing kit may include a protocol for freezing the cells. The protocol describes the procedure and conditions for freezing cells using the cell freezing composition. The cell freezing kit may further include a protocol for thawing the cells.

[0075] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention. Below, examples of reference forms are added. 1. A medium; antifreeze agent, Dissolved hydrogen, A composition for freezing cells, comprising: 2. The cell freezing composition according to 1., A composition for freezing cells, comprising hydrogen water. 3. A composition for freezing cells according to 1. or 2., A composition for cell freezing, wherein the dissolved hydrogen concentration of the composition for cell freezing is 0.1 ppm or more and 10.0 ppm or less when measured under atmospheric pressure at 25°C. 4. A composition for freezing cells according to any one of 1. to 3., A composition for cell freezing, wherein the oxidation-reduction potential of the composition for cell freezing is -700 mV or more and -100 mV or less when measured at atmospheric pressure and 25°C. 5. A composition for freezing cells according to any one of 1. to 4., A composition for cell freezing, wherein the pH of the composition for cell freezing is 6.8 or more and 8.5 or less when measured at atmospheric pressure and 25°C. 6. A composition for freezing cells according to any one of 1. to 5., A composition for cell freezing, wherein the dissolved oxygen concentration of the composition for cell freezing is 5.0 ppm or less when measured under atmospheric pressure at 25°C. 7. A composition for freezing cells according to any one of 1. to 6., A composition for freezing cells, wherein the cryoprotectant comprises one or more selected from the group consisting of dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol (EG), and glycerol. 8. A composition for freezing cells according to any one of 1. to 7., A composition for cell freezing, wherein the cells used for cell freezing include animal cells. 9. A culture medium; antifreeze agent, A composition for freezing cells, comprising: The redox potential of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is -700 mV or more and -100 mV or less; The pH of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is 6.8 or more and 8.5 or less. Composition for freezing cells. 10. Preparing a mixture of cells and a composition for cell freezing, the composition comprising a culture medium, a cryoprotectant, and dissolved hydrogen; freezing the mixture to obtain a frozen product; A method for freezing cells, comprising: 11. The cell freezing method according to 10, further comprising: The method for preparing the composition for cell freezing is a cell freezing method using at least one of the following methods: mixing hydrogen water with the culture medium, mixing hydrogen water with the cryoprotectant, adding hydrogen water to a mixture containing the culture medium and the cryoprotectant, adding hydrogen gas to the culture medium, adding hydrogen gas to the cryoprotectant, and adding hydrogen gas to a mixture containing the culture medium and the cryoprotectant. 12. A step of thawing the frozen product obtained by the cell freezing method described in 10. or 11. to obtain a thawed product; Culturing the cells contained in the resulting lysate; A cell culture method comprising: 13. A cell freezing kit including a storage container containing hydrogen water. 14. The cell freezing kit according to 13, A cell freezing kit, wherein the storage container contains a mixture of at least the hydrogen water and a culture medium. 15. A cell freezing kit according to 13. or 14., A cell freezing kit including a protocol for freezing cells.

[0076] Below, examples of reference forms are added. 1. A medium; antifreeze agent, Hydrogen water and A composition for freezing cells, comprising: 2. The cell freezing composition according to 1., A composition for cell freezing, wherein the oxidation-reduction potential of the composition for cell freezing is -700 mV or more and -100 mV or less when measured at atmospheric pressure and 25°C. 3. A composition for freezing cells according to 1. or 2., A composition for cell freezing, wherein the dissolved hydrogen concentration of the composition for cell freezing is 0.1 ppm or more and 1.6 ppm or less when measured under atmospheric pressure at 25°C. 4. A composition for freezing cells according to any one of 1. to 3., A composition for cell freezing, wherein the pH of the composition for cell freezing is 6.8 or more and 8.5 or less when measured at atmospheric pressure and 25°C. 5. A composition for freezing cells according to any one of 1. to 4., A composition for cell freezing, wherein the dissolved oxygen concentration of the composition for cell freezing is 5.0 ppm or less when measured under atmospheric pressure at 25°C. 6. A composition for freezing cells according to any one of 1. to 5., A composition for freezing cells, wherein the cryoprotectant comprises one or more selected from the group consisting of dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol (EG), and glycerol. 7. A composition for freezing cells according to any one of 1. to 6., A composition for cell freezing, wherein the cells used for cell freezing include animal cells. 8. A culture medium; antifreeze agent, A composition for freezing cells, comprising: The redox potential of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is -700 mV or more and -100 mV or less; The pH of the composition for cell freezing, when measured at atmospheric pressure and 25°C, is 6.8 or more and 8.5 or less. Composition for freezing cells. 9. A step of preparing a mixture of a composition for cell freezing according to any one of 1. to 8. and cells; freezing the mixture to obtain a frozen product; A method for freezing cells, comprising: 10. A step of thawing the frozen product obtained by the cell freezing method described in 9. to obtain a thawed product; Culturing the cells contained in the resulting lysate; A cell culture method comprising: 11. A cell freezing kit including a storage container containing hydrogen water. 12. The cell freezing kit according to 11, A cell freezing kit, wherein the storage container contains a mixture of at least the hydrogen water and a culture medium. 13. A cell freezing kit according to 11. or 12., A cell freezing kit including a protocol for freezing cells. [Example]

[0077] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0078] <Preparing hydrogen water> (Hydrogen water A) Using a hydrogen water generator (IDEC, Ultra Fine GALF, pressure dissolution method), hydrogen water A was prepared using distilled water at an ambient temperature of 25°C. The distilled water used had a dissolved hydrogen concentration of 0.00 ppm and a dissolved oxygen concentration of 8 to 9 ppm. Immediately after preparation, hydrogen water A had a dissolved hydrogen concentration of 1.5 ppm, a dissolved oxygen concentration of 2 ppm, a pH of 7.42, and an ORP of -476 mV. In addition, there was almost no change in the dissolved hydrogen concentration, dissolved oxygen concentration, pH, and ORP values ​​of hydrogen water A immediately after preparation and 15 minutes later.

[0079] The dissolved hydrogen concentration was measured at atmospheric pressure and 25°C using a dissolved hydrogen meter (H2 Microsensor, manufactured by Unisense). Dissolved oxygen was measured at atmospheric pressure and 25°C using a dissolved oxygen meter (Central Scientific Co., Ltd., CGS-5). The pH and ORP were measured at atmospheric pressure and 25°C using a pH / ORP meter (Toko Chemical Research Institute, TPX-999Si).

[0080] (Hydrogen water B) The hydrogen water A obtained immediately after preparation was sealed in an aluminum bag (GL Sciences, CCK-1), and the bag was filled with hydrogen gas and stored at 25°C under atmospheric pressure for 2 to 4 weeks. The hydrogen water B was used immediately after removal from the aluminum bag. After storage, immediately after opening, hydrogen water B had a dissolved hydrogen concentration of 1.5 ppm, a dissolved oxygen concentration of 2 ppm, a pH of 7.43, and an ORP of -525 mV.

[0081] <Preparation of cell freezing composition, cell cryopreservation, and thawing> Example 1 (1) Preparation of cell freezing composition Mouse-derived cells (KAC, MC3T3-E1, adherent cells) were added at 5 × 10 5 The cells were suspended in a culture medium (α-MEM medium) to a concentration of 1000 cells / mL, and a cell suspension was prepared in a 15 mL sterilized conical tube. Freshly prepared hydrogen water A was added to 100 μL of the obtained cell solution to adjust the dissolved hydrogen concentration, and then sterilized α-MEM medium (manufactured by Gibco, product name: Mem alpha basic (powder)) was mixed with this to obtain 900 μL of a mixture having the dissolved hydrogen concentration shown in Table 1 in a sterilized 15 ml conical tube. 700 μL of the resulting mixture, 200 μL of FCS (fetal calf serum, manufactured by GE Healthcare, product name: Hyclone Fetal Bovine Serum), and 100 μL of DMSO (dimethyl sulfoxide, manufactured by Nacalai Tesque, cryoprotectant) were placed in a 1 mL cryotube and mixed to prepare a pre-freezing cell preservation solution (composition for cell freezing). Similarly, six microtubes filled with a cell suspension containing cells in the cell preservation solution were prepared.

[0082] (2) Cell freezing operation Each cryotube was cooled to -80°C at a rate of -1°C / min using a cell freezing container (Mr. Frosty, manufactured by Cosmo Bio Co., Ltd.), and then frozen in a storage container containing liquid nitrogen by cooling to below -150°C. After confirming that the tubes were frozen, each microtube was stored in the storage container containing liquid nitrogen for 2 weeks.

[0083] (3) Cell thawing procedure Each cryotube was removed from the storage container and placed in a 37°C incubator for 3 minutes to confirm that the cell storage solution had thawed.

[0084] (4) Activity and proliferation evaluation The cell suspension containing cells frozen, preserved, and thawed in the cell preservation solution by the above steps (1) to (3) was used as the sample to be evaluated. Six samples were obtained from each of the six cryotubes. Each of the obtained samples was evaluated by the MTT assay described below, and changes in cell proliferation over 7 days after thawing were assessed.

[0085] The above operations were carried out under atmospheric pressure at 25°C unless otherwise specified. The dissolved hydrogen concentration, pH, and ORP were measured for each of the mixed solution immediately after preparation, the pre-freezing cell preservation solution immediately after preparation, and the post-freezing cell preservation solution immediately after thawing. The results are shown in Table 1.

[0086] (Comparative Example 1) A sample was obtained in the same manner as in Example 1, except that sterilized water was used as the mixed solution instead of hydrogen water A, and a mixed solution of the sterilized water and α-MEM medium was used. The sterilized water used had a dissolved hydrogen concentration of 0.00 ppm and a dissolved oxygen concentration of 8 to 9 ppm.

[0087] Example 2 A sample was obtained in the same manner as in Example 1, except that hydrogen water B stored in an aluminum bag was used instead of hydrogen water A.

[0088] [Table 1]

[0089] <MTTアッセイ> The MTT activity per cell number was measured according to the following procedure. Using the samples to be measured obtained in the examples and comparative examples, 5.0 × 10 cells were 5 cells / mL ~ 1.5 × 10 6 Culture medium (α-MEM medium) was added to a concentration of 100 cells / mL, and the mixture was mixed to prepare a solution. 100 μL of the mixed solution was seeded into 96 wells of a 96-well plate (manufactured by Corning) and cultured for 30 minutes in a CO 2 incubator at 37° C. and 5% carbon dioxide concentration. A mixed solution of WST-8 and 1-Methoxy PMS was added to each well in an amount of 10 μL. Subsequently, the cells were cultured for 60 minutes in a CO2 incubator at 37°C and a carbon dioxide concentration of 5%. After the incubation, the absorbance at 450 nm was measured using a microplate reader (Molecular Devices, product name: SpectraMax i3). The absorbance value obtained was used as the cell viability (MTT activity) of cell metabolism (respiration). The MTT activity of six samples was measured in the same manner. The absorbance thus obtained was divided by the number of cells used to calculate the MTT activity per cell number. Figure 1 shows the MTT activity per cell number (average value of six samples) for Example 1 and Comparative Example 1. The cell count was measured using a hemocytometer.

[0090] <Cell proliferation> Using the samples to be measured obtained in the Examples and Comparative Examples, cells were cultured according to the following procedure, and the number of cells was measured over time up to 7 days after culture. FIG. 2 shows cell growth curves showing the relationship between the number of cells (average value of six samples) and the number of days of culture in Example 1 and Comparative Example 1. The thawed cell stock solution was transferred to a conical tube and centrifuged at 1,000 rpm for 3 minutes, and the supernatant was discarded. 5 The cells were suspended in a culture medium (α-MEM medium) at a concentration of 100 cells / mL. The cell suspension was mixed with 10 mL of the culture medium in a separately prepared culture dish having a diameter of 100 mm. The cells were then cultured in a CO2 incubator at 37°C and 5% carbon dioxide. After culturing, the cells were detached from the culture dish and collected. The number of collected cells was counted using a hemocytometer.

[0091] By using the cell freezing composition containing hydrogen water of Example 1, the MTT activity value of the cells after freezing and thawing was higher and the cell proliferation rate was increased compared to Comparative Example 1. Example 2 also showed a similar trend to Example 1. Therefore, it was found that the cell proliferation rate after thawing was increased for cells cryopreserved using the cell freezing composition, regardless of whether or not hydrogen water in Examples 1 and 2 was prepared immediately before use.

[0092] (Examples 3 to 5, Comparative Example 2) (1') Preparation of cell freezing composition Mouse-derived cells (KAC, MC3T3-E1, adherent cells) were added at 5 × 10 5 The cells were suspended in a culture medium (α-MEM medium) to a concentration of 1000 cells / mL, and a cell suspension was prepared in a 15 mL sterilized conical tube. Separately, hydrogen gas was dissolved under pressure in α-MEM medium to prepare a hydrogen-containing medium. To 100 μL of the obtained cell solution, a hydrogen-containing medium and a normal α-MEM medium were added to obtain a mixed solution adjusted to a predetermined dissolved hydrogen concentration.

[0093] In Examples 3 and 4, hydrogen gas was added to the medium under atmospheric pressure to prepare a hydrogen-containing medium. In Example 5, the medium was sealed in an aluminum bag (GL Sciences, CCK-1), pressurized to 0.04 to 0.2 MPa, hydrogen gas was added, and the medium was shaken for 3 to 5 minutes or more to prepare a hydrogen-containing medium in which the dissolved hydrogen was supersaturated. In Comparative Example 2, hydrogen gas was not added, that is, a hydrogen-containing medium was not used.

[0094] 700 μL of the resulting mixture, 200 μL of FCS (fetal calf serum, manufactured by GE Healthcare, product name: Hyclone Fetal Bovine Serum), and 100 μL of DMSO (dimethyl sulfoxide, manufactured by Nacalai Tesque, cryoprotectant) were placed in a 1 mL cryotube and mixed to prepare a pre-freezing cell preservation solution (cell freezing composition) having the dissolved hydrogen concentration shown in Table 2. Similarly, six microtubes filled with a cell suspension containing cells in a cell preservation solution were prepared.

[0095] The dissolved hydrogen concentration was measured at atmospheric pressure and 25°C using a dissolved hydrogen meter (H2 Microsensor, manufactured by Unisense).

[0096] (2) Cell freezing operation Each cryotube was cooled to -80°C at a rate of -1°C / min using a cell freezing container (Mr. Frosty, manufactured by Cosmo Bio Co., Ltd.), and then frozen in a storage container containing liquid nitrogen by cooling to below -150°C. After confirming that the tubes were frozen, each microtube was stored in the storage container containing liquid nitrogen for 2 weeks.

[0097] (3) Cell thawing procedure Each cryotube was removed from the storage container and placed in a 37°C incubator for 3 minutes to confirm that the cell storage solution had thawed.

[0098] (4) Activity and proliferation evaluation The cell suspension containing cells frozen, preserved, and thawed in the cell preservation solution by the above steps (1') to (3) was used as the sample to be evaluated. Six samples were obtained from each of the six cryotubes. In Comparative Example 2, a sample (reference example) was also prepared in which the freezing steps (2) and (3) above were not carried out. For each sample obtained,<MTTアッセイ> The results are shown in Table 3 and Figure 3. The MTT activity value per cell count for the "non-frozen" sample (reference example) was normalized to 1.00.

[0099] [Table 2]

[0100] The cell cryopreservation methods using the cell freezing compositions of Examples 3 to 5 resulted in higher MTT activity values ​​of the cells after freezing and thawing compared to Comparative Example 2.

[0101] Even when mouse-derived cells (RAW 264.7) or human-derived cells (THP-1) were used instead of mouse-derived cells (MC3T3-E1), the results showed that the MTT activity values ​​of the cells after freeze-thawing were higher in Examples 1 and 2 than in Comparative Example 1, and in Examples 3 to 5 than in Comparative Example 2.

[0102] This application claims priority based on Japanese Patent Application No. 2019-185161, filed on October 8, 2019, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A culture medium; antifreeze agent, Dissolved hydrogen, A composition for freezing cells, comprising: A composition for cell freezing, wherein the dissolved oxygen concentration of the composition for cell freezing is 5.0 ppm or less when measured at atmospheric pressure and 25°C.

2. 2. The cell freezing composition according to claim 1, A composition for freezing cells, comprising hydrogen water.

3. 3. The cell freezing composition according to claim 1 or 2, A composition for cell freezing, wherein the dissolved hydrogen concentration of the composition for cell freezing is 0.1 ppm or more and 10.0 ppm or less when measured at atmospheric pressure and 25°C.

4. The composition for cell freezing according to any one of claims 1 to 3, A composition for cell freezing, wherein the oxidation-reduction potential of the composition for cell freezing is -700 mV or more and -100 mV or less when measured at atmospheric pressure and 25°C.

5. The cell freezing composition according to any one of claims 1 to 4, A composition for cell freezing, wherein the pH of the composition for cell freezing is 6.8 or more and 8.5 or less when measured at atmospheric pressure and 25°C.

6. The composition for freezing cells according to any one of claims 1 to 5, A composition for freezing cells, wherein the cryoprotectant comprises one or more selected from the group consisting of dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol (EG), and glycerol.

7. The composition for cell freezing according to any one of claims 1 to 6, A composition for cell freezing, wherein the cells used for cell freezing comprise invertebrate-derived cells, mammal-derived cells, amphibian-derived cells, reptile-derived cells, or fish-derived cells.

8. The composition for cell freezing according to any one of claims 1 to 7, The cells used for cell freezing are: ES cells, iPS cells, Mesenchymal cells, fibroblasts, muscle cells, bone cells, adipocytes, nerve cells, epithelial cells, Mesenchymal stem cells, fibroblast stem cells, muscle stem cells, bone stem cells, adipose stem cells, neural stem cells, epithelial stem cells, A composition for freezing cells, comprising cells induced to differentiate from ES cells, cells induced to differentiate from iPS cells, cells induced to differentiate from mesenchymal stem cells, cells induced to differentiate from fibroblast stem cells, cells induced to differentiate from muscle stem cells, cells induced to differentiate from osteostem cells, cells induced to differentiate from adipose stem cells, cells induced to differentiate from neural stem cells, or cells induced to differentiate from epithelial stem cells.

9. The composition for freezing cells according to any one of claims 1 to 8, A composition for freezing cells, comprising serum or an additive.

10. A method for producing a composition for freezing cells, comprising a culture medium, a cryoprotectant, and dissolved hydrogen, using at least one of the following methods: mixing hydrogen water with the culture medium, adding hydrogen water to the cryoprotectant, adding hydrogen water to a mixed solution containing the culture medium and the cryoprotectant, adding hydrogen gas to the culture medium, adding hydrogen gas to the cryoprotectant, and adding hydrogen gas to a mixed solution containing the culture medium and the cryoprotectant (however, the addition of hydrogen gas is carried out in a container under pressure); A method for producing a composition for cell freezing, comprising the step of preparing the composition for cell freezing, wherein the dissolved oxygen concentration when measured at atmospheric pressure and 25°C is 5.0 ppm or less.

11. preparing a mixture of cells and a composition for cell freezing, the composition comprising a culture medium, a cryoprotectant, and dissolved hydrogen; and freezing the mixture to obtain a frozen product. A method for freezing cells, wherein in the preparing step, the dissolved oxygen concentration of the composition for freezing cells is 5.0 ppm or less when measured at atmospheric pressure and 25°C.

12. The cell freezing method according to claim 11, The method for freezing cells, wherein the cells comprise invertebrate-derived cells, mammal-derived cells, amphibian-derived cells, reptile-derived cells, or fish-derived cells.

13. 13. The cell freezing method according to claim 12, The method for preparing the composition for cell freezing is a cell freezing method using at least one of the following methods: mixing hydrogen water with the culture medium, mixing hydrogen water with the cryoprotectant, adding hydrogen water to a mixture containing the culture medium and the cryoprotectant, adding hydrogen gas to the culture medium, adding hydrogen gas to the cryoprotectant, and adding hydrogen gas to a mixture containing the culture medium and the cryoprotectant.

14. A step of thawing the frozen product obtained by the cell freezing method according to claim 12 or 13 to obtain a thawed product; Culturing the cells contained in the resulting lysate; A cell culture method comprising:

15. A storage container containing hydrogen water having a dissolved oxygen concentration of 5.0 ppm or less when measured at atmospheric pressure and 25°C is provided, The storage container contains the hydrogen water in a liquid phase and hydrogen gas in a gas phase.

16. 16. The cell freezing kit according to claim 15, A kit for freezing cells, including a medium or a cryoprotectant.

17. 17. The cell freezing kit according to claim 16, A cell freezing kit, wherein the storage container contains a mixture of at least the hydrogen water and a culture medium.

18. 18. The cell freezing kit according to claim 16 or 17, A cell freezing kit including a protocol for freezing cells.

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